Development of a specific enzyme linked immunosorbent assay ...

Development of a specific enzyme linked immunosorbentassay (ELISA) for the detection of fluoroquinolone antibioticresidues in chicken liver, prawn and milk

Author:Zahid, Muhammad

Publication Date:2011

DOI:https://doi.org/10.26190/unsworks/15516

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Development of a Specific Enzyme Linked Immunosorbent Assay

(ELISA) for the Detection of Fluoroquinolone Antibiotic Residues

in Chicken Liver, Prawn and Milk

Muhammad Zahid

Thesis submitted in partial fulfilment of the requirement

for the Degree of Master of Science (Research)

School of Chemical Engineering

The University of New South Wales

April, 2011

i

ABSTRACT

Extensive utilisation of fluoroquinolones (FQs) in agricultural and aquacultural practices

leads to two major food safety issues: 1) the issues regarding the presence of FQs

residues in food and 2) the development of FQs resistant bacteria in animals, which may

be transferable to humans. This may have an implication to human health, in particular

for the treatment of infection.

This thesis describes the design and synthesis of novel haptens for (enrofloxacin) ENR,

ciprofloxacin and norfloxacin, the production of specific antibodies, and the formatting

and characterizing of an indirect competitive Enzyme‐Linked ImmunoSorbent Assay (ELISA)

for detection of ENR. The design and synthesis of FQs haptens involved the following

approaches: 1) synthesising ENR hapten by attaching a tert‐butyl linker on a carboxylic

group, and 2) synthesising ciprofloxacin and norfloxacin haptens by attaching a 4‐

bromobutane NHS ester and bromocrotyl NHS ester linkers respectively on the

piperazinyl moiety.

Highly specific polyclonal antibodies were generated against the ENR‐Keyhole Limpet

Haemocyanine (KLH) conjugate. The optimized ELISA exhibited higher sensitivity in a

homologous assay than a heterologous assay, suggesting the developed antibody was

extremely specific to ENR. The ELISA displayed an IC50 value of 11.7 µg L‐1 ± 1.7 with a limit

of detection (LOD) value of 2.4 µg L‐1 ± 0.4. High specificity of the developed assay was

evidenced by low cross‐reactivity to seven structurally related FQs compounds

(danofloxacin, enofloxacin, sarafloxacin, perfloxacin, nalidixic acid, ciprofloxacin and

norfloxacin). The effects of surfactants (Tween 20), water miscible organic solvent

(methanol, ethanol, acetonitrile, and acetone) and pH conditions (5.5‐9.5) were also

evaluated. Briefly, Tween 20 affected considerably on colour development, but not the

assay sensitivity. Of the solvents tested, up to 5% methanol showed no significant effects

on the assay sensitivity. The sample preparation were also optimized for milk, chicken

liver and prawn, yielding the recoveries between 64 (± 3) and 125 (± 8)%.

This ELISA will be particularly useful for screening ENR residues in animal and marine

derived products to improve antibiotic safety in developing countries such as Indonesia.

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ACKNOWLEDGEMENTS

“In the name of ALLAH, the most gracious and merciful”

First and foremost I would like to express my sincerest gratitude to ALLAH SWT, my Lord

and Cherisher, for guiding and blessing in every single step of my life. Indeed, without his

help and will, nothing is accomplished.

I am heartily thankful to my supervisor, Dr. Nanju Alice Lee, whose encouragement,

guidance and support from the very early to the final stage of this research, enabled me

to develop an understanding of the subject as well as gave me extraordinary experiences

throughout the work. Her encouragement has triggered and nourished my intellectual

maturity that I will benefit from. I am grateful in every possible way and hope to keep up

our collaboration in the future.

I gratefully acknowledge my co‐supervisor, A/Prof. Naresh Kumar for his advice, expertise,

and supervision. It has been an honour to have had the opportunity to work in his

laboratory. I also would like to express my gratitude to Dr. George Iskander for his

involvement, ideas, research passion and crucial contribution has made him one of the

backbones of this research.

Many thanks go to Dr. Victor Wong for the precious time rendered in proofreading this

thesis, including the critical comments and scientific ideas forwarded. I would like to

thank Camillo Toraborelli for his technical assistance in the laboratory and his kindness in

putting every requested chemical on my bench.

Special thanks also to all fellow researchers in the Food Science and Nutrition Research

laboratory, School of Chemical Engineering; Eriyanto Yusnawan, Maria Veronica Hoie,

Karrie Kam, Yang Lu, Kim‐Yen Phan‐Thien, Ebtihal Khodijah, Chatchaporn Uraipong and

Norma Karim, as well as those who are affiliated with, the Organic Chemistry laboratory,

School of Chemistry; Samuel Kutty, Hakan Kandemir, Kitty Ho, Ren Chen, Rick Zhang,

Raymond Chen, Adeline Lukmantara and Asep Kurnia Permana, for their support,

knowledge, sharing, laughs, and even tears. I am so grateful to have you guys who are

always around. You are such wonderful people and always make our laboratory such an

incredible place to be.

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For financial assistance, I would like to express my deepest gratitude to AusAID through

Australian Development Scholarship (ADS) for giving me a great opportunity to study at

The University of New South Wales, Australia. Without this support, this research project

would have been impossible. I would like to record my gratitude to the Indonesian

government, and more specifically, The National Veterinary Drug Assay Laboratory, The

Ministry of Agriculture of Republic of Indonesia, for allowing me to improve my skills and

knowledge through this research project in Australia.

This research project would not have been possible without the support of numerous

other people, so I offer my regards and blessings to all who supported me in any way for

the duration of the project.

Lastly, I would like to dedicate this thesis to my beloved families and especially for my

beloved wife; Isnindar, my little angel; Maura Thalita Chairinniswa, for their prayers,

patience, understanding and endless love, throughout the duration of my studies.

iv

ABBREVIATIONS

Ab‐ENR1 polyclonal antibody of ENR

Amax maximum absorbance

APCI atmospheric pressure chemical ionization

BSA bovine serum albumin

cBSA cationised‐bovine serum albumin

CBT checkerboard titration

CDCl3 deuterated chloroform

CIP ciprofloxacin

CIP1‐OA a conjugate of ciprofloxacin N‐hydroxysuccinimide ester and ovalbumin

CIP2‐OA a conjugate of ciprofloxacin butyl N‐hydroxysuccinimide ester and ovalbumin

CMC 1‐cyclohexyl‐3‐(2‐morphplinyl‐4‐ethyl) carbodiimide methyl p‐toluene sulfonate

CNS central nervous system

CV coefficient of variation

D2O deuterium oxide

DAD diode array detection

DAN danofloxacin

DCC dicyclohexylcarbodiimide

DCM dichloromethane

DEPT distortionless enhancement by polarization transfer

DIC diisopropylcarbodiimide

DMAP 4‐dimethylaminopyridine

DMF dimethylformamide

DMSO dimethylsulfoxide

DNA deoxyribonucleic acid

EDC 1‐ethyl‐3‐(3‐dimethyl‐aminopropyl) carbodiimide hydrochloride

v

ELISA enzyme linked immunosorbent assay

ENO enoxacin

ENR enrofloxacin

ENR1‐KLH a conjugate of ENR N‐hydroxysuccinimide ester and keyhole limpet haemocyanin

ENR1‐OA a conjugate of ENR N‐hydroxysuccinimide ester and ovalbumin

ENR2‐OA conjugate of ENR acid and keyhole limpet haemocyanin

ESI electrospray ionization

EtOH ethanol

EU European Union

FAO/WHO Food and Agriculture Organisation/World Health Organisation

FCL full cream liquid milk

FCP full cream milk powder

FG fish gelatine

FLD fluorescence detection

FLU flumequine

FQs fluoroquinolones

FSANZ Food Standard Australia New Zealand

GAT gatifloxacin

GC/MS gas chromatography/mass spectrometry

GI gastrointestinal

HAS human albumin serum

HPLC high performance liquid chromatography

HRMS high resolution mass spectrometry

HRP horseradish peroxidise

IBCF isobutylchloroformate

Ig immunoglobulin

IgG immunoglobulin g

JEFCA joint expert committee on food additives

vi

KLH keyhole limpet haemocyanin

LC/MS liquid chromatography mass spectrometry

LC/MS‐MS liquid chromatography/tandem mass spectrometry

LOD limit of detection

LOQ limit of quantification

LRMS low resolution mass spectrometry

Lv1 chicken liver from an organic source

Lv2 chicken liver from Coles supermarket

Lv3 chicken liver from butcher

MAb monoclonal antibody

MAR marbofloxacin

MeOH methanol

MRLs maximum residue limits

NaH sodium hydride

NAL nalidixic acid

NAL‐OA a conjugate of nalidixic acid and ovalbumin

NHS N‐hydroxysuccinimide

NMR nuclear magnetic resonance

NOR norfloxacin

NOR‐OA a conjugate of norfloxacin and ovalbumin

NSAID non steroid anti inflammatory drug

OFL ofloxacin

OA ovalbumin

OXO oxolinic acid

PAb polyclonal antibody

PBS phosphate buffer saline

PEF pefloxacin

PEF‐OA a conjugate of pefloxacin ovalbumin

vii

Pr1 local prawn

Pr2 Thai prawn

Pr3 Malaysian prawn

Rf retardation factor

RO reverse osmosis

SAR sarafloxacin

SAR‐OA a conjugate of sarafloxacin and ovalbumin

SARs structure activity relationships

SD standard deviation

SKL skim milk liquid

SKP skim milk powder

TEA triethylamine

TFA tetrafluoroacetic acid

TLC thin layer chromatography

TMB 3,3’,5,5’‐tetramethylbenzidine

USP the U.S. of pharmacopeia

UV ultraviolet

viii

TABLE OF CONTENTS

ABSTRACT ....................................................................................................... I

ACKNOWLEDGEMENTS ..................................................................................... II

ABBREVIATIONS ............................................................................................. IV

LIST OF FIGURES ........................................................................................... XIII

LIST OF TABLES ............................................................................................. XX

CHAPTER 1. INTRODUCTION .............................................................................. 1

1.1BACKGROUND OF RESEARCH ................................................................................................. 1

1.2 METHOD DEVELOPMENT FOR FLUOROQUINOLONE RESIDUES ..................................................... 3

1.3 THE OBJECTIVES AND SIGNIFICANCE OF STUDY ......................................................................... 4

CHAPTER 2. LITERATURE REVIEW ........................................................................ 5

2.1. FLUOROQUINOLONE ANTIBACTERIAL AGENTS ......................................................................... 5

2.1.1. Overview of quinolone .......................................................................................... 5

Figure 2.1 Structure of 7‐chloro‐4‐quinoline (Nalidixic acid). ............................. 5

2.1.2. Chemical structure of fluoroquinolone antibiotics ............................................... 5

2.1.3. Generations of Quinolones ................................................................................... 7

2.1.4. Mechanism of action ............................................................................................ 9

2.1.5. Structure activity relationships of fluoroquinolones .......................................... 10

2.1.6. Clinical use in animal and human ....................................................................... 10

2.1.7 Fluoroquinolones used in this study .................................................................... 13

2.1.7.2. ENR .............................................................................................................. 14

2.1.7.3. Norfloxacin .................................................................................................. 15

2.1.8. Pharmacokinetics and toxicity ........................................................................... 16

2.1.7. Adverse effects and drug interactions ................................................................ 16

2.2. PUBLIC HEALTH CONCERNS ............................................................................................... 18

2.2.1. Food safety ......................................................................................................... 19

2.2.2. Maximum residue limits (MRLs) ......................................................................... 19

2.2.3. Fluoroquinolone resistant bacteria .................................................................... 22

2.3 ANALYTICAL METHODS FOR DETECTING OF FLUOROQUINOLONES ANTIBIOTIC RESIDUES .................. 23

2.3.1. Instrument‐based methods ................................................................................ 24

2.3.1.1. High performance liquid chromatography (HPLC) ...................................... 24

2.3.1.2. Liquid chromatography / mass spectrometry (LC/MS) and (LC‐MS/MS).... 25

2.3.1.3. Gas Chromatography / Mass Spectrometry (GC/MS) ................................. 25

2.3.2. Bioanalytical or immunochemical methods ....................................................... 27

2.3.2.1. Immunoaffinity chromatography (IAC) ....................................................... 27

ix

2.3.2.2. Biosensors/Immunosensors ........................................................................ 28

2.3.2.3. Immunoassays ............................................................................................. 28

2.4. ELISA (ENZYME‐LINKED IMMUNOSORBENT ASSAY) .............................................................. 30

2.4.1. Principle .............................................................................................................. 30

2.4.2 ELISA Format ....................................................................................................... 30

2.5. DEVELOPMENT OF IMMUNOASSAY FOR FLUOROQUINOLONE ANTIBIOTICS .................................. 36

2.5.1. Hapten design and synthesis .............................................................................. 36

2.5.1.1. Selection of spacer arm point attachment for fluoroquinolone antibiotics 37

2.5.1.2. Competing hapten to carrier protein ratio.................................................. 38

2.5.1.3. Conjugation methods .................................................................................. 39

2.5.1.3.1. Carboxylic groups .................................................................................. 40

2.5.1.3.2. Amine groups ........................................................................................ 42

2.5.2. Antibody production ........................................................................................... 43

2.5.2.1. Overview ...................................................................................................... 43

2.5.2.2. Polyclonal antibodies ................................................................................... 43

2.5.2.3. Monoclonal antibodies ................................................................................ 44

2.5.3. Immunoassay format ......................................................................................... 45

2.5.4. Assay characterization ....................................................................................... 46

2.5.4.1. Calibration curve ......................................................................................... 46

2.5.4.2. Sensitivity, limit of detection (LOD) and limit of quantification (LOQ) ....... 46

2.5.4.3. Specificity and cross reactivity .................................................................... 47

2.5.4.4. Matrix interference ..................................................................................... 48

2.5.4.5. Assay accuracy and precision ...................................................................... 49

2.6 CONCLUSION .................................................................................................................. 49

CHAPTER 3. HAPTEN SYNTHESIS ....................................................................... 51

3.1 INTRODUCTION ............................................................................................................... 51

3.2 MATERIALS AND INSTRUMENTATION ................................................................................... 53

3.2.1 Materials and chemicals ..................................................................................... 53

3.2.1.1 Materials ....................................................................................................... 53

3.2.1.2 Chemicals ...................................................................................................... 53

3.2.2 Equipment and instrumentation ......................................................................... 54

3.2.2.1 Thin Layer Chromatography (TLC) ................................................................ 54

3.2.2.2 Nuclear Magnetic Resonance (NMR) spectroscopy ..................................... 54

3.2.2.3 Mass Spectrometry ....................................................................................... 54

3.3 HAPTEN SYNTHESIS .......................................................................................................... 54

3.3.1 The attachment via carboxylic group of FQs as a spacer arm for ENR acid hapten

...................................................................................................................................... 55

3.3.1.1 Synthesis of tert‐butyl 3‐(1‐cyclopropyl‐7‐(4‐ethylpiperazin‐1‐yl)‐6‐fluoro‐4‐

oxo‐1,4‐dihydroquinoline‐3‐carboxamido)propanoate, [ENRtert‐butyl], compound

(1), scheme 1 ............................................................................................................ 55

x

3.3.1.2 Synthesis of 3‐(1‐cyclopropyl‐7‐(4‐ethylpiperazin‐1‐yl)‐6‐fluoro‐4‐oxo‐1,4‐

dihydroquinoline‐3‐carboxamido)propanoic acid, [ENR acid], compound (2),

scheme 2 ................................................................................................................... 55

3.3.2 The spacer arm attachment via the piperazinyl moiety of ciprofloxacin to create

ciprofloxacinbutyl NHS ester hapten ............................................................................ 57

3.3.2.1 Synthesis of 4‐bromobutane NHS ester linker, compound (3), scheme 3 ... 57

3.3.2.2 Synthesis of 1‐cyclopropyl‐7‐(4‐(4‐((2,5‐dioxopyrrolidin‐1‐

yl)oxy)butyl)piperazin‐1‐yl)‐6‐fluoro‐4‐oxo‐1,4‐dihydroquinoline‐3‐carboxylic acid,

[ciprofloxacin butyl NHS ester hapten], compound (4), scheme 4 .......................... 58

3.3.3 The spacer arm attachment via piperazinyl moiety of norfloxacin to form

norfloxacin crotyl NHS ester ......................................................................................... 59

3.3.3.1 Synthesis of 4‐bromocrotonic acid compound (5), scheme 5 ...................... 60

3.3.3.2 Synthesis of bromocrotyl NHS ester linker, compound (6), scheme 6 ........ 60

3.3.3.3 Synthesis of (E)‐7‐(4‐(4‐((2,5‐dioxopyrrolidin‐1‐yl)oxy)‐4‐oxobut‐2‐en‐1‐

yl)piperazin‐1‐yl)‐1‐ethyl‐6‐fluoro‐4‐oxo‐1,4‐dihydroquinoline‐3‐carboxylic acid

[Norfloxacincrotyl NHS ester hapten], compound (7), scheme 7 ............................ 60

3.4 RESULT AND DISCUSSION ................................................................................................... 62

3.4.1 Hapten selection and synthesis ........................................................................... 62

3.4.2 ENR acid hapten synthesis................................................................................... 63

3.4.2.1 Synthesis of tert‐butyl 3‐(1‐cyclopropyl‐7‐(4‐ethylpiperazin‐1‐yl)‐6‐fluoro‐4‐

oxo‐1,4‐dihydroquinoline‐3‐carboxamido)propanoate, ENR tert‐butyl .................. 63


dihydroquinoline‐3‐carboxamido)propanoic acid, ENR acid hapten ....................... 65

3.4.3 Ciprofloxacin bromobutane NHS ester hapten synthesis .................................... 65

3.4.3.1 Synthesis of (1‐(4‐bromobutoxy)pyrrolidine‐2,5‐dione), 4‐bromobutane

NHS ester linker ........................................................................................................ 66

3.4.3.2. Synthesis of (1‐cyclopropyl‐7‐(4‐(4‐((2,5‐dioxopyrrolidin‐1‐yl)oxy)butyl)

piperazin‐1‐yl)‐6‐fluoro‐4‐oxo‐1,4‐dihydroquinoline‐3‐carboxylic acid),

ciprofloxacin butane NHS ester hapten ................................................................... 66

3.4.4 Norfloxacin crotyl NHS ester hapten synthesis ................................................... 68

3.4.4.1 Synthesis of 4‐bromocrotonic acid ............................................................... 69

3.4.4.2 Synthesis of bromocrotyl NHS ester linker ................................................... 69

3.4.4.3 Synthesis of norfloxacin crotyl NHS ester hapten ........................................ 70

3.5 CONCLUSION .................................................................................................................. 70

CHAPTER 4. DEVELOPMENT OF THE SPECIFIC ENR ELISA (ENR‐ELISA) ........................ 71

4.1 INTRODUCTION ............................................................................................................... 71

4.2 MATERIALS AND METHODS ................................................................................................ 73

4.2.1 Materials and Instrumentation ........................................................................... 73

4.2.1.1 Materials ....................................................................................................... 73

4.2.1.2 Instruments .................................................................................................. 73

4.2.2 Antibody production and characterization ......................................................... 73

xi

4.2.2.1 Preparation of conjugates of hapten and carrier proteins or enzyme ........ 73

4.2.2.2 Immunisation and antibody production ...................................................... 74

4.2.2.3 Purification of Rabbit IgG ............................................................................. 75

4.2.2.4 Determining antibody concentration ........................................................... 75

4.2.2.5 Determining optimum working concentration by checkerboard titration .. 75

4.2.2.6 Determining Sensitivity ................................................................................ 76

4.2.2.6.1 Preparation of standard solution ........................................................... 76

4.2.2.6.2 Indirect Competitive ELISA protocol ...................................................... 76

4.2.2.6.3 Determination of standard curve parameter ........................................ 77

4.2.2.7 Optimisation of ENR ELISA conditions .......................................................... 78

4.2.2.7.1 The effect of antiserum diluents ........................................................... 78

4.2.2.7.2 The effect of organic solvents ................................................................ 78

4.2.2.7.3. The effect of buffer solutions (pH conditions) ..................................... 78

4.2.2.8 Determining specificity ................................................................................. 78

4.2.2.9 Study of Matrix Effects ................................................................................. 79

4.2.2.9.1 Animal and marine product samples ..................................................... 79

4.2.2.9.2 Protocol for sample extraction of chicken liver and prawn .................. 79

4.2.2.9.3 Protocol for matrix effect determination of milk .................................. 80

4.2.3 Spiking and recovery studies ............................................................................... 80

4.2.3.1 Protocol for spiking of chicken liver and prawn with ENR ........................... 80

4.2.3.2 Protocol for spiking of milk with ENR ........................................................... 80

4.3 RESULTS AND DISCUSSION ................................................................................................. 81

4.3.1 Antibody production and optimal concentration of ENR1 antiserum ................. 81

4.3.2 Antibody characterisation ................................................................................... 82

4.3.2 Assay Sensitivity .................................................................................................. 84

4.3.3 Evaluation of assay parameters (IC80, IC50, IC20 and maximum absorbance) ..... 85

4.3.4 Characteristics of ENR ELISA ................................................................................ 87

4.3.4.1 Assay specificity ............................................................................................ 87

4.3.4.2 Assay Optimisation ....................................................................................... 90

4.3.4.2.1 Effects of diluents .................................................................................. 91

4.3.4.2.2 Effects of organic solvents ................................................................ 93

4.3.4.2.3 Effect of pH ............................................................................................ 99

4.3.5 Matrix Interferences .......................................................................................... 100

4.3.5.1 Effect of matrix in milk ............................................................................... 101

4.3.5.2 Effect of matrix in chicken liver and shrimp samples ................................. 110

*Extraction solvent is 50 mM NaOH:MeOH:PBS=1:9:90. Each value represents

the mean of triplicates (n=3) with a standard deviation (SD). *no significant

difference with PBS. ^significant difference with extraction solvent (control)

......................................................................................................................... 111

4.3.6 Recovery studies ................................................................................................ 122

4.3.7 Linear regression of spiking and recoveries ...................................................... 126

CHAPTER 5. CONCLUSIONS AND RECOMMENDATIONS ....................................... 130

xii

5.1 CONCLUSIONS ............................................................................................................... 130

5.2. RECOMMENDATIONS ..................................................................................................... 132

REFERENCES ............................................................................................... 133

xiii

LIST OF FIGURES

FIGURE 2.1 STRUCTURE OF 7‐CHLORO‐4‐QUINOLINE (NALIDIXIC ACID)...............................5

FIGURE 2.2 GENERAL CHEMICAL STRUCTURE OF A FQ.........................................................6

FIGURE 2.3 CHEMICAL STRUCTURE OF CIPROFLOXACIN.....................................................13

FIGURE 2.4 CHEMICAL STRUCTURE OF ENR........................................................................14

FIGURE 2.5 CHEMICAL STRUCTURE OF NORFLOXACIN.......................................................15

FIGURE 2.6 SCHEMATIC PRESENTATION OF AN INDIRECT COMPETITIVE ELISA.................32

FIGURE 2.7 AN IMMUNOGEN IS MADE BY COUPLING A HAPTEN WITH A CARRIER

MOLECULE USING A CONJUGATION REAGENT...................................................................37

FIGURE 2.8 REACTION OF A CARBOXYLIC ACID WITH A CHLORFORMATE FORMS AN

AMINE‐REACTIVE MIXED ANHYDRIDE.................................................................................41

FIGURE 2.9 REACTION OF A CARBOXYLIC ACID WITH CARBODIIMIDE FORMS AN O‐

ACYLISOUREA......................................................................................................................42

FIGURE 2.10 REACTION OF A CARBOXYLIC ACID GROUP WITH CARBODIIMIDE‐NHS FORMS

AN ACTIVE‐SUCCINIMIDE ESTER.........................................................................................43

FIGURE 3.1 FQ DRUGS USED IN SYNTHESISING HAPTENS...................................................53

FIGURE 3.2 POINT OF THE ATTACHMENT OF LINKERS ON FQ ANTIBIOTICS.......................54

FIGURE 3.3 THE CHEMICAL STRUCTURE OF TERT‐BUTYL ENR HAPTEN……………………………65

FIGURE 3.4 THE CHEMICAL STRUCTURE OF ENR ACID HAPTEN..........................................67

FIGURE 3.5 THE CHEMICAL STRUCTURE OF 4‐BROMOBUTANE NHS ESTER LINKER...........68

FIGURE 3.6 THE CHEMICAL STRUCTURE OF CIPROFLOXACIN BUTANE NHS ETHER

HAPTEN...............................................................................................................................68

FIGURE 3.7 SYNTHESIS OF CIPROFLOXACIN BUTANE NHS ETHER HAPTEN CATALYSED BY

TEA......................................................................................................................................69

FIGURE 3.8 SYNTHESIS OF CIPROFLOXACIN BROMO NHS ETHER HAPTEN CATALYSED BY

K2CO3...................................................................................................................................70

FIGURE 3.9 THE CHEMICAL STRUCTURE OF 4‐BROMOCROTONIC ACID.............................71

FIGURE 3.10 THE CHEMICAL STRUCTURE BROMOCROTYL NHS ESTER...............................71

FIGURE 4.1 THE SCHEMATIC REACTION OF ENR HAPTEN‐PROTEIN CONJUGATION...........76

FIGURE 4.2 SCHEMATIC PRESENTATION OF AN INDIRECT COMPETITIVE ELISA FOR

ENR......................................................................................................................................79

FIGURE 4.3 TITRATION CURVE OF ABENR1‐KLH AGAINST ENR1‐OA FROM SIX DIFFERENT

xiv

BLEEDS (FIRST BLEED TO SIXTH BLEED) BY AN INDIRECT ELISA FORMAT............................84

FIGURE 4.4 TITRATION CURVES OF ABENR1‐KLH#1 (FROM BLEED#1) AGAINST THE EIGHT

HAPTEN‐PROTEIN CONJUGATES (PEF‐OA, SAR‐OA, ENR2‐OA, NOR‐OA, NAL‐OA, CIP2‐OA,

CIP1‐OA AND ENR1‐OA).....................................................................................................85

FIGURE 4.5 CALIBRATION CURVES FOR ABENR1‐KLH (AVERAGE OF 25 ANALYSES) BASED

ON THE ABSORBANCE () VS ENR CONCENTRATION AND THE % INHIBITION () VS ENR

CONCENTRATION USING THE OPTIMISED CONCENTRATIONS OF ANTI‐ENR ANTIBODIES

AND IMMOBILISED ENRANTIGENWITH AN IC50 VALUE OF 11.8µG L‐1 ± 1.7 AND LOD 2.4 µG

L‐1± 0.4. ± REPRESENTS STANDARD DEVIATION……………………………………………………………….88

FIGURE 4.6 %CV OF THE ABSORBANCE () AND THE % INHIBITION () BASED ON AN

AVERAGE OF 25 ANALYSES.................................................................................................89

FIGURE 4.7 PLOT OF IC20 (), IC50 () AND IC80 () VALUES OF THE 25 STANDARD CURVES..

THE MIDDLE SOLID LINES INDICATE THE AVERAGE VALUES OF IC20, IC50 AND IC80. THE

DOTTED LINES INDICATE THE UPPER AND LOWER LIMITS OF STANDARD DEVIATION;

SD........................................................................................................................................89

FIGURE 4.8 EFFECTS OF DILUENTS (PBS, 1% FG‐PBS, 1% FG‐PBS + 0.05% TWEEN 20 AND 1%

FG‐PBS + 0.1% TWEEN 20) ON COLOUR DEVELOPMENT OF THE ELISA BASED ON

ABENR1‐KLH. EACH VALUE REPRESENTS A MEAN OF TRIPLICATES (N=3) WITH A

STANDARD DEVIATION (SD) VALUE OF PBS, 1% FG‐PBS, 1% FG‐PBS + 0.05% TWEEN 20

AND 1% FG‐PBS + 0.1% TWEEN 20 WAS 0.01, 0.02, 0.02 AND 0.01, RESPECTIVELY.

STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST ( = 0.05)...................................94

FIGURE 4.9 STANDARD CURVES OF ENR IN DIFFERENT DILUENTS (PBS, 1% FG‐PBS, 1% FG‐

PBS + 0.05% TWEEN 20 AND 1% FG‐PBS + 0.1% TWEEN 20). EACH VALUE REPRESENTS THE

MEAN OF TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD) VALUE OF PBS, 1% FG‐

PBS, 1% FG‐PBS + 0.05% TWEEN 20 AND 1% FG‐PBS + 0.1% TWEEN 20 WAS 1.5, 3.4, 0.8

AND 0.7, RESPECTIVELY. STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST ( =

0.05)....................................................................................................................................95

FIGURE 4.10 EFFECTS OF METHANOL (5% MEOH, 10% MEOH AND 20% MEOH) ON

COLOUR DEVELOPMENT OF THE ENR1‐ELISA. EACH VALUE REPRESENTS THE MEAN OF

TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD) VALUE OF PBS, 5% MEOH, 10%

MEOH AND 20% MEOH WAS 0.01, 0.01, 0.02 AND 0.01, RESPECTIVELY. STATISTICAL

ANALYSIS WAS CALCULATED USING T‐TEST (Α = 0.05).......................................................96

xv

FIGURE 4.11 STANDARD CURVES OF ENR DISSOLVED IN DIFFERENT CONCENTRATIONS OF

METHANOL (5% MEOH, 10% MEOH AND 20% MEOH). EACH VALUE REPRESENTS THE

MEAN OF TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD) VALUE OF PBS, 5%

MEOH, 10% MEOH AND 20% MEOH WAS 1.1, 0.9, 4.7 AND 0.8, RESPECTIVELY.

STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST (Α = 0.05)...................................96

FIGURE 4.12 EFFECTS OF ACETONITRILE (5% ACETONITRILE, 10% ACETONITRILE AND 20%

ACETONITRILE) ON COLOUR DEVELOPMENT. EACH VALUE REPRESENTS THE MEAN OF

TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD) VALUE OF PBS, 5% ACETONITRILE,

10% ACETONITRILE AND 20% ACETONITRILE WAS 0.03, 0.03, 0.03 AND 0.02, RESPECTIVELY.

STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST (Α = 0.05)..................................97

FIGURE 4.13 STANDARD CURVES OF ENR IN DIFFERENT CONCENTRATIONS OF

ACETONITRILE (5% ACETONITRILE, 10% ACETONITRILE AND 20% ACETONITRILE). EACH

VALUE REPRESENTS THE MEAN OF TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD)

OF PBS, 5% ACETONITRILE, 10% ACETONITRILE AND 20% ACETONITRILE WAS 0.2, 12.4, 2.2

AND 3.6, RESPECTIVELY. STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST (Α =

0.05)....................................................................................................................................97

FIGURE 4.14 EFFECTS OF ACETONE (5% ACETONE, 10% ACETONE AND 20% ACETONE) ON

COLOUR DEVELOPMENT. EACH VALUE REPRESENTS THE MEAN OF TRIPLICATES (N=3)

WITH A STANDARD DEVIATION (SD) OF PBS, 5% ACETONE, 10% ACETONE AND 20%

ACETONE WAS 0.07, 0.03, 0.02 AND 0.04, RESPECTIVELY. STATISTICAL ANALYSIS WAS

CALCULATED USING T‐TEST (Α = 0.05)................................................................................98

FIGURE 4.15 STANDARD CURVES OF ENR IN DIFFERENT CONCENTRATIONS OF ACETONE (5%

ACETONE, 10% ACETONE AND 20% ACETONE). EACH VALUE REPRESENTS THE MEAN OF

TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD) OF PBS, 5% ACETONE, 10%

ACETONE AND 20% ACETONE WAS 2.2, 3.7, 1.8 AND 5.1, RESPECTIVELY. STATISTICAL

ANALYSIS WAS CALCULATED USING T‐TEST (Α = 0.05)........................................................98

FIGURE 4.16 EFFECTS OF ETHANOL (5% ETHANOL, 10% ETHANOL AND 20% ETHANOL) ON

COLOUR DEVELOPMENT OF THE ENR1‐ELISA. EACH VALUE REPRESENTS THE MEAN OF

TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD) OF PBS, ETOH 5%, ETOH 10% AND

ETOH 20% WAS 0.03, 0.01, 0.01 AND 0.03, RESPECTIVELY. STATISTICAL ANALYSIS WAS

CALCULATED USING T‐TEST (Α = 0.05)................................................................................99

xvi

FIGURE 4.17 STANDARD CURVES OF ENR IN DIFFERENT CONCENTRATIONS OF ETHANOL (5%

ETHANOL, 10% ETHANOL AND 20% ETHANOL) FOR ENR1‐ELISA AND EACH VALUE

REPRESENTS THE MEAN OF TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD) OF PBS,

ETOH 5%, ETOH 10% AND ETOH 20% WAS 1.9, 4.1, 2.5 AND 3.4, RESPECTIVELY.

STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST (Α = 0.05)...................................99

FIGURE 4.18 EFFECTS OF THE PH ON THE ENR1‐ELISA. THE CIRCLE INDICATES

ABSORBANCE AND THE TRIANGLE INDICATES IC50 VALUES AGAINST PH AND EACH VALUE

REPRESENTS A MEAN OF TRIPLICATES (N=3) OF PH 5.5, 6.5, 7.5, 8.5 AND 9.5 WITH A

STANDARD DEVIATION (SD) VALUE WAS 4.5, 0.8, 0.1, 4.5 AND 0.6, RESPECTIVELY.

STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST (Α = 0.05).................................102

FIGURE 4.19 EFFECTS OF SKIM MILK LIQUID (SKL), DILUTED 1:5, 1:10 AND 1:20 WITH PBS

ON COLOUR DEVELOPMENT. EACH VALUE REPRESENTS THE MEAN OF TRIPLICATES (N=3)

WITH A STANDARD DEVIATION (SD) VALUE OF PBS, DILUTED SKL 1:5, 1:10 AND 1:20 WITH

PBS WAS 0.01, 0.04, 0.01 AND 0.04, RESPECTIVELY. STATISTICAL ANALYSIS WAS

CALCULATES USING T‐TEST (Α = 0.05).............................................................................104

FIGURE 4.20 STANDARD CURVES OF ENR DISSOLVED IN SKIM MILK LIQUID (SKL), DILUTED

1:5, 1:10 AND 1:20 WITH PBS.EACH VALUE REPRESENTS THE MEAN OF TRIPLICATES (N=3)

WITH STANDARD DEVIATION (SD) VALUE OF PBS, DILUTED SKL 1:5, 1:10 AND 1:20 WITH

PBS WAS 0.8, 1.7, 1.2 AND 0.6, RESPECTIVELY. STATISTICAL ANALYSIS WAS CALCULATED

USING T‐TEST (Α = 0.05)....................................................................................................104

FIGURE 4.21 EFFECTS OF SKIM MILK POWDER (SKP), DILUTED 1:5, 1:10 AND 1:20 WITH

PBS, ON COLOUR DEVELOPMENT OF THE ENR1‐ELISA. EACH VALUE REPRESENTS THE

MEAN OF TRIPLICATES (N=3) WITH STANDARD DEVIATION (SD) OF PBS, DILUTED SKP 1:5,

1:10 AND 1:20 WITH PBS WAS 0.01, 0.04, 0.01 AND 0.04, RESPECTIVELY. STATISTICAL

ANALYSIS WAS CALCULATED USING T‐TEST (Α = 0.05)......................................................105

FIGURE 4.22 STANDARD CURVES OF ENR DISSOLVED IN SKIM MILK POWDER (SKP), 1:5,

1:10 AND 1:20 WITH PBS. EACH VALUE REPRESENTS THE MEAN OF TRIPLICATES (N=3)

WITH A STANDARD DEVIATION (SD) OF PBS, DILUTED SKP 1:5, 1:10 AND 1:20 WITH PBS

WAS 0.7, 1.6, 1.2 AND 0.6, RESPECTIVELY. STATISTICAL ANALYSIS WAS CALCULATED

USING T‐TEST (Α = 0.05)...................................................................................................105

FIGURE 4.23 EFFECTS OF FULL CREAM MILK LIQUID (FCL), DILUTED 1:5, 1:10 AND 1:20

WITH PBS ON COLOUR DEVELOPMENT OF THE ENR1‐ELISA. EACH VALUE REPRESENTS THE

xvii

MEAN OF TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD) OF PBS, DILUTED FCL 1:5,

1:10 AND 1:20 WITH PBS WAS 0.01, 0.04, 0.01 AND 0.04, RESPECTIVELY. STATISTICAL

ANALYSIS WAS CALCULATED USING T‐TEST (Α = 0.05).....................................................106

FIGURE 4.24 STANDARD CURVES OF ENR DISSOLVED IN FULL CREAM MILK LIQUID (FCL),

DILUTED 1:5, 1:10 AND 1:20. EACH VALUE REPRESENTS THE MEAN OF TRIPLICATES (N=3)

WITH A STANDARD DEVIATION (SD) OF PBS, DILUTED FCL 1:5, 1:10 AND 1:20 WITH PBS

WAS 0.8, 1.7, 1.2 AND 0.6, RESPECTIVELY. STATISTICAL ANALYSIS WAS CALCULATED

USING T‐TEST (Α = 0.05)....................................................................................................106

FIGURE 4.25 EFFECTS OF FULL CREAM MILK POWDER (FCP), DILUTED 1:5, 1:10 AND 1:20

WITH PBS ON COLOUR DEVELOPMENT. EACH VALUE REPRESENTS THE MEAN OF

TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD) OF PBS, DILUTED FCP 1:5, 1:10

AND 1:20 WITH PBS WAS 0.01, 0.04, 0.01 AND 0.04, RESPECTIVELY. STATISTICAL ANALYSIS

WAS CALCULATED USING T‐TEST (Α = 0.05)......................................................................107

FIGURE 4.26 STANDARD CURVES OF ENR DISSOLVED IN FULL CREAM MILK POWDER (FCP),

DILUTED 1:5, 1:10 AND 1:20 WITH PBS. EACH VALUE REPRESENTS THE MEAN OF

TRIPLICATES (N=3) WITH A STANDARD DEVIATION (SD) OF PBS, DILUTED FCP 1:5, 1:10

AND 1:20 WITH PBS WAS 1.1, 1.2, 0.9, 1.3, RESPECTIVELY. STATISTICAL ANALYSIS WAS

CALCULATED USING T‐TEST (Α = 0.05)..............................................................................107

FIGURE 4.27 EFFECTS OF SKIM MILK LIQUID (SKL) AND SKIM MILK POWDER (SKP),

DILUTED 1:10 WITH PBS, ON COLOUR DEVELOPMENT OF THE ENR1‐ELISA. EACH VALUE

REPRESENTS THE MEAN OF FIVE REPLICATES (N=5) WITH A STANDARD DEVIATION (SD)

VALUE OF PBS, DILUTED SKL AND SKP 1:10 WITH PBS WAS 0.1, 0.2 AND 0.2, RESPECTIVELY.


FIGURE 4.28 STANDARD CURVES OF ENR IN SKIM MILK LIQUID (SKL) AND SKIM MILK

POWDER (SKP), DILUTED 1:10 WITH PBS. EACH VALUE REPRESENTS THE MEAN OF FIVE

REPLICATES (N=5) WITH A STANDARD DEVIATION (SD) VALUE OF PBS, DILUTED SKL AND

SKP 1:10 WITH PBS WAS 1.2, 2.5 AND 2.8, RESPECTIVELY. STATISTICAL ANALYSIS WAS


FIGURE 4.29 EFFECT OF FULL CREAM MILK LIQUID (FCL) AND FULL CREAM MILK POWDER

(FCP), DILUTED 1:10 WITH PBS, ON COLOUR DEVELOPMENT. EACH VALUE REPRESENTS

THE MEAN OF FIVE REPLICATES (N=5) WITH A STANDARD DEVIATION (SD) OF PBS,

xviii

DILUTED SKL AND SKP 1:10 WITH PBS WAS 0.1, 0.2 AND 0.1, RESPECTIVELY. STATISTICAL


FIGURE 4.30 STANDARD CURVES OF ENR DISSOLVED IN FULL CREAM MILK LIQUID (FCL)

AND FULL CREAM MILK POWDER (FCP), DILUTED 1:10 WITH PBS. EACH VALUE

REPRESENTS THE MEAN OF FIVE REPLICATES (N=5) WITH A STANDARD DEVIATION (SD) OF

PBS, DILUTED SKL AND SKP 1:10 WITH PBS WAS 1.6, 2.0 AND 2.0, RESPECTIVELY.


FIGURE 4.31 EFFECTS OF CHICKEN LIVER (COLES, LV2), DILUTED 1:20 WITH PBS, ON

COLOUR DEVELOPMENT. EACH VALUE REPRESENTS THE MEAN OF TRIPLICATES (N=3)

WITH A STANDARD DEVIATION (SD) VALUE OF PBS, EXTRACTION SOLVENT AND

EXTRACTED LIVER WAS 0.03, 0.04 AND 0.03, RESPECTIVELY. STATISTICAL ANALYSIS WAS


FIGURE 4.32 STANDARD CURVES OF ENR DISSOLVED IN CHICKEN LIVER EXTRACT (COLES,

LV2). EACH VALUE REPRESENTS THE MEAN OF TRIPLICATES (N=3) WITH A STANDARD

DEVIATION (SD) VALUE OF PBS, EXTRACTION SOLVENT AND EXTRACTED LIVER WAS 0.8,

0.9 AND 0.6, RESPECTIVELY. STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST (Α =

0.05)..................................................................................................................................114

FIGURE 4.33 EFFECTS OF LOCAL PRAWN EXTRACT (PR1) ON COLOUR DEVELOPMENT.

EACH VALUE REPRESENTS THE MEAN OF TRIPLICATES (N=3) WITH A STANDARD


0.01 AND 0.01, RESPECTIVELY. STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST (Α

= 0.05)...............................................................................................................................114

FIGURE 4.34 STANDARD CURVES OF ENR DISSOLVED IN THE LOCAL PRAWN EXTRACT

(PR1). EACH VALUE REPRESENTS THE MEAN OF TRIPLICATES (N=3) WITH A STANDARD


3.1 AND 1.8, RESPECTIVELY. STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST (Α =

0.05)..................................................................................................................................115

FIGURE 4.35 EFFECTS OF ORGANIC CHICKEN LIVER (LV1), DILUTED 1:50 WITH PBS, ON

COLOUR DEVELOPMENT. EACH VALUE REPRESENTS THE MEAN OF FIVE REPLICATES (N=5)

WITH A STANDARD DEVIATION (SD) VALUE OF PBS, EXTRACTION SOLVENT, AND

EXTRACTED ORGANIC CHICKEN LIVER (LV1) WAS 0.2, 0.1 AND 0.2, RESPECTIVELY.


xix

FIGURE 4.36 STANDARD CURVES OF ENR DISSOLVED IN ORGANIC CHICKEN LIVER EXTRACT

(LV1). EACH VALUE REPRESENTS THE MEAN OF REPLICATES (N=5) WITH A STANDARD

DEVIATION (SD) VALUE OF PBS, EXTRACTION SOLVENT, AND EXTRACTED ORGANIC

CHICKEN LIVER (LV1) WAS 2.2, 0.8 AND 0.8, RESPECTIVELY. STATISTICAL ANALYSIS WAS


FIGURE 4.37 EFFECTS OF CHICKEN LIVER EXTRACT (LV2), DILUTED 1:50 WITH PBS, ON


WITH A STANDARD DEVIATION (SD) OF PBS, EXTRACTION SOLVENT, AND EXTRACTED


CALCULATED USING T‐TEST (Α = 0.05)............................................................................118

FIGURE 4.38 STANDARD CURVES OF ENR DISSOLVED IN CHICKEN LIVER EXTRACT (LV2),

DILUTED 1:50 WITH PBS. EACH VALUE REPRESENTS THE MEAN OF FIVE REPLICATES (N=5)




FIGURE 4.39 EFFECTS OF CHICKEN LIVER EXTRACT (LV3), DILUTED 1:50 WITH PBS, ON





FIGURE 4.40 STANDARD CURVES OF ENR DISSOLVED INA CHICKEN LIVER EXTRACT

(LV3).EACH VALUE REPRESENTS THE MEAN OF FIVE REPLICATES (N=5) WITH A STANDARD

DEVIATION (SD) OF PBS, EXTRACTION SOLVENT, AND EXTRACTED CHICKEN LIVER (LV3)

WAS 5.4, 4.7 AND 3.5, RESPECTIVELY. STATISTICAL ANALYSIS WAS CALCULATED USING T‐

TEST (Α = 0.05)...................................................................................................................119

FIGURE 4.41 EFFECTS OF LOCAL PRAWN EXTRACT (PR1), DILUTED 1:50 WITH PBS, ON



PRAWN (PR1) WAS 0.2, 0.2 AND 0.2, RESPECTIVELY. STATISTICAL ANALYSIS WAS


FIGURE 4.42 STANDARD CURVES OF ENR IN LOCAL PRAWN EXTRACT (PR1). EACH VALUE

REPRESENTS THE MEAN OF FIVE REPLICATES (N=5) WITH A STANDARD DEVIATION (SD) OF

xx

PBS, EXTRACTION SOLVENT, AND EXTRACTED LOCAL PRAWN (PR1) WAS 6.3, 2.7 AND 3.6,

RESPECTIVELY. STATISTICAL ANALYSIS WAS CALCULATED USING T‐TEST (Α =

0.05)..................................................................................................................................120

FIGURE 4.43 EFFECTS OF PRAWN FROM COLES SUPERMARKET (PR2), EXTRACT DILUTED

1:50 WITH PBS ON COLOUR DEVELOPMENT. EACH VALUE REPRESENTS THE MEAN OF FIVE

REPLICATES (N=5) WITH A STANDARD DEVIATION (SD) PBS, EXTRACTION SOLVENT, AND

EXTRACTED LOCAL PRAWN (PR2) WAS 0.1, 0.1 AND 0.1 RESPECTIVELY. STATISTICAL


FIGURE 4.44 STANDARD CURVES OF ENR IN AN EXTRACT FROM THE PRAWN PURCHASED

FROM COLES SUPERMARKET (PR2). EACH VALUE REPRESENTS THE MEAN OF FIVE

REPLICATES (N=5) WITH A STANDARD DEVIATION (SD) PBS, EXTRACTION SOLVENT, AND

EXTRACTED PRAWN (PR2) WAS 2.2, 1.6 AND 2.5, RESPECTIVELY. STATISTICAL ANALYSIS

WAS CALCULATED USING T‐TEST (Α = 0.05)......................................................................121

FIGURE 4.45 EFFECTS OF EXTRACT FROM THE PRAWN PURCHASED FROM A LOCAL

BUTCHER (PR3) ON COLOUR DEVELOPMENT. EACH VALUE REPRESENTS THE MEAN OF

FIVE REPLICATES (N=5) WITH A STANDARD DEVIATION (SD) PBS, EXTRACTION SOLVENT,

AND EXTRACTED PRAWN (PR3) WAS 0.1, 0.1 AND 0.1, RESPECTIVELY. STATISTICAL


FIGURE 4.46 STANDARD CURVES OF ENR IN AN EXTRACT FROM THE PRAWN PURCHASED

FROM A LOCAL BUTCHER (PR3). EACH VALUE REPRESENTS THE MEAN OF FIVE REPLICATES

(N=5) WITH A STANDARD DEVIATION (SD) PBS, EXTRACTION SOLVENT, AND EXTRACTED

PRAWN (PR3) WAS 4.4, 1.3 AND 2.3, RESPECTIVELY. STATISTICAL ANALYSIS WAS


FIGURE 4.47 CORRELATION BETWEEN THE LEVELS OF ENR SPIKING IN SKIM MILK LIQUID

(SKL), SKIM MILK POWDER (SKP), FULL CREAM MILK LIQUID (SKL), FULL CREAM MILK

POWDER (SKP) AND ESTIMATES BY THE ENR1‐ELISA. AVERAGE VALUES (µG L‐1) OF

SPIKING AND SPIKING LEVEL (µG L‐1) FROM IN PBS BUFFER.............................................127

FIGURE 4.48 CORRELATION BETWEEN THE LEVELS OF ENR SPIKING IN SKIM MILK LIQUID

(SKL), SKIM MILK POWDER (SKP), FULL CREAM MILK LIQUID (SKL), FULL CREAM MILK

POWDER (SKP) AND ESTIMATES BY THE ENR1‐ELISA. AVERAGE VALUES (µG L‐1) OF

SPIKING AND SPIKING LEVEL (µG L‐1) FROM IN DILUTED SAMPLES...................................128

xxi

FIGURE 4.49 CORRELATION BETWEEN THE LEVELS OF ENR SPIKING IN ORGANIC CHICKEN

LIVER (LV1), CHICKEN LIVER FORM COLES (LV2), CHICKEN LIVER FROM BUTCHER (LV3),

LOCAL PRAWN (PR1), THAI PRAWN (PR2) AND MALAYSIAN PRAWN (PR3) AND ESTIMATES

BY THE ENR1‐ELISA. AVERAGE VALUES (µG L‐1) OF SPIKING AND SPIKING LEVEL (µG L‐1)

FROM IN EXTRACTION SOLVENT.......................................................................................128

FIGURE 4.50 CORRELATION BETWEEN THE LEVELS OF ENR SPIKING IN ORGANIC CHICKEN

LIVER (LV1), CHICKEN LIVER FORM COLES (LV2), CHICKEN LIVER FROM BUTCHER (LV3),

LOCAL PRAWN (PR1), THAI PRAWN (PR2) AND MALAYSIAN PRAWN (PR3) AND ESTIMATES

BY THE ENR1‐ELISA. AVERAGE VALUES (µG L‐1) OF SPIKING AND SPIKING LEVEL (µG L‐1)

FROM IN EXTRACTED SAMPLES.........................................................................................129

xxii

LIST OF TABLES

TABLE 2.1 GENERATIONS OF FQS..........................................................................................7

TABLE 2.1 GENERATIONS OF FQS (CONTINUED)...................................................................8

TABLE 2.2 FQ ANTIBIOTIC USED IN VETERINARY MEDICINES................................................8

TABLE 2.3 OLDER FQS MARKETED FOR BOTH HUMAN AND VETERINARY USES.................10

TABLE 2.4 MODERN FLUOROQUINOLONES MARKETED FOR BOTH HUMAN AND

VETERINARY USES...............................................................................................................11

TABLE 2.5 PROPOSED AND/OR APPROVED DOSAGES OF VARIOUS FQS USED IN

VETERINARY MEDICINE.......................................................................................................12

TABLE 2.6 COMMON ADVERSE REACTIONS ASSOCIATED WITH SOME FQS.......................16

TABLE 2.7 MRL VALUES ESTABLISHED BY THE EU AND JECFA FOR RESIDUES OF FQS AND

QUINOLONES USED IN VETERINARY MEDICINE……………………………………………………………….20


QUINOLONES USED IN VETERINARY MEDICINE(CONTINUED)...........................................21


QUINOLONES USED IN VETERINARY MEDICINE(CONTINUED)............................................22

TABLE 2.8 ELISAS DEVELOPED FOR FQ ANTIBIOTICS...........................................................32

TABLE 2.8 ELISAS DEVELOPED FOR FQ ANTIBIOTICS (CONTINUED)....................................33





TABLE 4.1 THE CONCENTRATIONS OF ENR USED TO SPIKE SAMPLES.................................83

TABLE 4.2 THE IC50 VALUES (µG L‐1) OF THE ASSAYS BASED ON THE COMBINATION OF 14

FQ HAPTEN‐OA CONJUGATES COMPETED WITH ENR FOR ABENR1‐KLH BY INDIRECT COMPETITIVE ELISA.............................................................................................................86

TABLE 4.3 STANDARD CURVE PARAMETERS AND PRECISION OF ENR ASSAY......................88

TABLE 4.4 THE IC50 (µG L‐1) AND CROSS REACTIVITY (%CR) FOR FQS RELATED

COMPOUNDS.......................................................................................................................91

xxiii

TABLE 4.4 THE IC50 (µG L‐1) AND CROSS REACTIVITY (%CR) FOR FQS RELATED COMPOUNDS

(CONTINUED)......................................................................................................................92

TABLE 4.5 EFFECT OF DILUENTS ON ANTIBODY’S SENSITIVITY...........................................93

TABLE 4.6 EFFECTS OF WATER MISCIBLE ORGANIC SOLVENTS ON THE PERFORMANCE OF

THE ENR ELISA...................................................................................................................100

TABLE 4.7 EFFECTS OF MILK MATRIX ON COLOUR DEVELOPMENT (AMAX) AND ASSAY

SENSITIVITY (IC50)..............................................................................................................108

TABLE 4.8 MATRIX EFFECTS OF PRE‐TREATED MILK, DILUTED 1:10 WITH PBS, ON COLOUR

DEVELOPMENT (AMAX) AND ASSAY SENSITIVITY (IC50)......................................................111

TABLE 4.9 EFFECTS OF CHICKEN LIVER (LV2) AND PRAWN (PR1) SAMPLES, IN A 20‐FOLD

DILUTION WITH PBS ON COLOUR DEVELOPMENT (AMAX) AND ASSAY SENSITIVITY

(IC50)..................................................................................................................................112

TABLE 4.10 EFFECTS OF CHICKEN LIVER AND PRAWN EXTRACTS, ON COLOUR

DEVELOPMENT (AMAX) AND ASSAY SENSITIVITY (IC50).......................................................115

TABLE 4.11 % RECOVERIES ENR SPIKED IN MILK AS DETECTED BY ELISA..........................124

TABLE 4.12 %RECOVERIES OF ENR SPIKED IN CHICKEN LIVER AS DETECTED BY

ELISA..................................................................................................................................125

TABLE 4.13 % RECOVERIES OF ENR SPIKED IN PRAWN AS DETECTED BY

ELISA..................................................................................................................................126

1

CHAPTER 1. INTRODUCTION

1.1 Background of research

An antibiotic is defined as any substance produced by a microorganism that inhibits or kills

other microorganisms, primarily bacteria (Klotins, 2005). Antibiotics can also be produced

synthetically. The main function of antibiotics is for treatment of diseases, as well as

prophylaxis to prevent illness before the development of clinical signs (Smith et al., 1999).

Fluoroquinolones (FQs) belong to a class of synthetic antibiotics that have broad‐spectrum

biological mechanisms for the treatment and prevention of a wide range of bacterial

infections in both humans and veterinary animals. They act through the inhibition of

essential bacterial enzymes, namely DNA gyrase and topoisomerase IV, by interfering the

DNA rejoining reaction (Huet et al., 2006). FQs are highly effective against most Gram‐

negative bacteria, mycoplasma, and some Gram‐positive bacteria, but are less effective

against the Streptococci group and obligate anaerobic bacteria (Brown, 1996).

ENR, ciprofloxacin, and norfloxacin are the most frequently used FQs in veterinary

medicines, particularly in poultry, and are also often administered to large animals, such as

cattle and pigs, as well as pets, cats and dogs. According to the sample entry data of the

pharmaceutical products from the National Veterinary Drug Assay Laboratory, the Ministry

of Agriculture, Gunungsindur‐Bogor, West Java Province, Indonesia, FQs totaled 30% of the

pharmaceutical products used between 2004 and 2007. ENR has the largest number of

brand names among the single active ingredient products. Currently there are about 54

brand names of ENR being distributed in Indonesian. The Ministry of Agriculture of

Indonesia also has approved ENR for use in aquaculture since 2005.

Recently, there are about 44 generic names of quinolones and FQs are being distributed

worldwide for use as human and animal medicines, with some being banned, such as

trovafloaxcin, grepafloxacin, clinafloxacin and gatifloxacin, due to severe adverse effects in

humans and animals (i.e. crystalluria, lethal hepatic damage, cardiovascular disorder,

2

hypoglycaemia) (Bertino and Fish, 2000, Neu, 1992, Sárközy, 2001). Ciprofloxacin,

danofloxacin, difloxacin, marbofloxacin, orbifloxacin, norfloxacin, ofloxacin, levofloxacin

and moxifloxacin are amongst FQs still approved for clinical uses in veterinary and humans

in Canada, USA and European countries (Mandell and Tillotson, 2002), and are listed in the

U.S. of Pharmacopeia USP(2007).

In Australia, none of these compounds are permitted for use in aquaculture without a

specific permit or prescription. According to the Australia and New Zealand Food Standards

Code (FSANZ), quinolones and FQs residues must not be detectable in any animal‐derived

foods for human consumption. Since oxolinic acid is still regularly used in veterinary

medicine, a maximum residue limits (MRLs) of 0.01 mg kg‐1 is permitted in pacific salmon.

The newest memberof this family is orbifloxacin which is currently registered for use in cats

and dogs (Johnston et al., 2002).

Even though FQs have been effective in controlling various infections in the agriculture and

aquaculture industries, administrating these drugs above the levels recommended, or

using intensively for a long period, could lead to accumulation of FQ residues in animal

products. Prolong exposure, and hencebioaccumulation of FQ residues in livestock

products has been suggested as a potential route for the development of antibiotic

resistant bacteria in humans, leading to increase in treatment failure (Huet et al., 2006).

Subsequently, the U.S. Food Drug Administration (FDA) had banned the use of ENR in

poultry soon after the emergence of FQs‐resistant Campylobacter species in both poultry

and humans was discovered (Zhao et al., 2009).

To minimise the risk of human exposure FQthrough food consumption, and to regulate FQ

residues in marine and animal‐derived products to safe levels, it is crucial to establish MRLs.

The regulatory authorities in U.S. (FDA, 2005), European (European Union, 1990), Japanese

(Ministry of Agriculture, Forestry and Fishery, 2000) and Chinese (Ministry of Agriculture,

2003) have established MRLs for ENR, ciprofloxacin and their metabolites between 30 and

300 µg kg‐1 in marine and animal derived products (Brás Gomes et al., 2010). Meanwhile,

the National Standardization Agency of Indonesia still refers to FAO/WHO Expert

3

Committee on Food Authority (JECFA) for guidance in establishing MRLs for FQ residues in

Indonesia. The MRLs for ENR, ciprofloxacin and their active metabolites have been set at

between 100 µg kg‐1 and 300 µg kg‐1 in milk, muscle and edible tissues, in Indonesia. While

much lower MRL of 30 µg kg‐1for ENR and its metabolites is set by the European

commission, Regulation 2377/90 (Volmer et al., 1997), causing some conflicts for

international trade.

1.2 Method Development for Fluoroquinolone Residues

Generally, there are two main analytical techniques used to identify and quantify antibiotic

residues in food, namely conventional methods using instrumentation and immunoassay

methods based on antigen‐antibody binding interaction. Instrument‐based methods

include high performance liquid chromatography (HPLC) with UV/fluorescence detection

(Carlucci, 1998, Yorke and Froc, 2000, Espinosa‐Mansilla et al., 2006,Hung, 2007,), liquid

chromatography‐mass spectrometry (LC/MS) (Marchesini et al., 2007), liquid

chromatography‐tandem mass spectrometry (LC‐MS/MS) (Johnston et al., 2002, Bogialli et

al., 2008), capillary electrophoresis (Fierens et al., 2000) gas chromatography (GC)

(Takatsuki, 1991) and thin layer chromatography (TLC) and high performance thin layer

chromatography (HPTLC) (Choma et al., 2004, Gaugin and Abjean, 1998).

Although the instrument‐based methods are very sensitive and highly accurate, they are

also laborious often involving extensive sample preparation and sample interference

removing steps. Hence, these methods are not sufficiently cost‐effective, are technically

complex, and requires highly skilled operators, in many instances not suited to analytical

environment of the developing countries. In view of the above, the immunoassay method

is preferred as complement to instrument‐based methods, since it is simple and faster,

with high sample throughput for a single analyte, shows high sensitivity, high specificity,

greater cost‐effectiveness, require little training on the technique, available in convenient

kits for analyses of specific compounds and for families of compounds (Toldra and Reig,

2006).

4

A variety of immunoassay methods have been successfully developed for FQ residues:

ciprofloxacin (Duan and Yuan, 2001), sarafloxacin (Holtzapple et al., 1997, Huet et al.,

2006), ENR(Watanabe H. et al., 2002, Hammer and Heeschen, 1995, Bucknall et al., 2003),

norfloxacin (Bucknall et al., 2003, Huet et al., 2006), flumequine (Coillie et al., 2004),

pefloxacin (Lu et al., 2006), marbofloxacin (Sheng et al., 2009a), gatifloxacin (Zhao et al.,

2007), nalidixic acid (Bucknall et al., 2003), danofloxacin (Sheng et al., 2009b) and ofloxacin

(Sun et al., 2007). Some researchers have also attempted to develop generic assay or class

specific assays to detect FQ multi‐residues in a single test (Wang et al., 2007a, Li et al.,

2008, Huet et al., 2006, Tittlemier et al., 2008, Kato et al., 2007). Others have developed

compound specific assays that are useful as a quantitative analytical tool (Bucknall et al.,

2003, Coillie et al., 2004, Zhao et al., 2007, Lu et al., 2006).

1.3 The objectives and significance of study

This research project, therefore, aims to improve the sensitivity and specificity of

antibodies for the detection FQ residues in marine and animal‐derived products through

novel hapten design and synthesis. Since immunoassay provides low operating costs, this

method is suitable to be developed and applied as a complement to instrument‐based

method for detecting FQs residues in food, particularly in developing countries, such as

Indonesia.

5

CHAPTER 2. LITERATURE REVIEW

2.1. Fluoroquinolone antibacterial agents

2.1.1. Overview of quinolone

George Lesher and his coworkers discovered the first quinolone, nalidixic acid, in 1962.

Since then it had been medically important as an antimicrobial agent for the treatment of

urinary tract infections in humans. Nowadays a wide range of antibiotics has been derived

based on the core unit of the quinolone structure (Wagman and Wentland, 2007). For

example, nalidixic acid, as shown on Figure 2.1, was derived from a recrystallisation of 7‐

chloro‐4‐quinoline during the synthesis of chloroquine which was used to treat malaria

during World War II. The majority of quionolones in clinical use belong to a subset of

fluoroquinolones (FQs), which have a fluorine atom attached to the central ring system,

typically at the C‐6 position or C‐7 position.

Figure 2.1 Structure of 7‐chloro‐4‐quinoline (Nalidixic acid).

Several quinolone antibiotics, such as oxolinic acid, cinoxacin and pipemidic acid,

introduced in the 1970s, had a narrow spectrum of antibacterial activity, frequent

incidence of adverse effects, poor tissue penetration and distribution, and subsequent

inadequate serum concentration. Further development work led to a more powerful class

of agents known as FQs. Norfloxacin was the first FQs synthesized, which exhibited better

spectrum of bacterial activity (Wagman and Wentland, 2007).

2.1.2. Chemical structure of fluoroquinolone antibiotics

The 6‐fluoroquinolones are also known as 4‐quinolones or quinolones and are derived

from or related to nalidixic acid and oxolinic acid. Some substitutions on the FQ structural

backbone (Figure 2.2), such as the R1 substitutions, are usually alkyl groups (e.g.

6

cyclopropyl, ethyl, fluorethyl, methylamino), fluorophenyl group, thiazine or oxazine ring.

The R2substitutions are often piperazine derivatives (piperazin‐1‐yl, 4‐methylpiperazin‐1‐yl,

3‐methylpiperazin‐1‐yl) and X substitutions are either carbon or nitrogen atom (Figure 2.2).

Figure 2.2 General chemical structure of a FQ.

The basic backbone of FQ has the following features: A nitrogen atom at position 1 on the

bicyclic aromatic ring structure, a carboxylic acid group at position 3 is important for

antimicrobial activity. A fluorine atom at position 6 enhances the efficacy and spectrum

activity against Gram negative and positive bacterial pathogens (Brown, 1996). Moreover,

the carboxylic group at position 3 renders these compounds acidic, whereas the 7‐

piperazinylquinolones include additional amine groups which are basic. Therefore, the 7‐

piperazinylquinolones in an aqueous solution may be present as three different species, i.e.

cationic, zwitterionic and anionic, while the other FQs and quinolones are either neutral or

anionic (Hernandez‐Arteseros et al., 2002).

FQs and quinolones with a piperazinyl moiety have two pKa values: pKa1≈6 and pKa2≈9, thus

they are always charged. They exist mostly in cationic forms at acidic pH, anionic forms at

basic pH and as zwitterionic at neutral pH. The carboxylic acid group have a pKa of ≈6 and

present as a neutral compound at acidic pH and as an anionic form at neutral and basic pH

(Yorke and Froc, 2000). Most FQs are highly soluble in both acidic and alkaline aqueous

solutions. Water solubility at physiological pH varies widely across these compounds,

depending on the substitutions on the FQs or quinolones’ carboxylic acid nucleus. Salt

forms of the FQs are freely soluble and are generally stable in aqueous solution (Brown,

1996).

7

2.1.3. Generations of Quinolones

The quinolones are divided and grouped based on features such as antibacterial spectrum

(either narrow or broad spectrum activity), fluorinated compounds (is known as

fluoroquinolones; FQs), the methods employed (to synthesise and develop new

generation), patent dates, specific decade (i.e. 60s, 70s, 80s etc.) and the structural

modification (to enhance biological and pharmacological activities) (Blondeau, 2004).

However, there is no standard used to determine which drugs belong to which generation.

Generally, the first generation drugs exhibit narrower spectrum activity than the later ones.

Nowadays, the first generation drug is rarely used due to severe toxicity. For instance,

nalidixic acid was the first quinolone drug listed as a carcinogen in 1998, including some

other generations being banned from clinical practice due to the same reason. The most

frequently prescribed drugs today are moxifloxacin, ciprofloxacin, levofloxacin and some

generic equivalents (Oliphant and Green, 2002). Table 2.1 shows FQ antibiotics have been

produced based on their generations and Table 2.2 presents FQ antibiotics used in

veterinary medicines (Oliphant and Green, 2002, Mella M et al., 2000, King et al., 2000,

Beneš, 2005, Ball, 2000).

Table 2.1 Generations of FQs

Generation Drugs Status

First generation Cinoxacin (Cinobac®)

Flumequine (Flubactin®)

Nalidixic acid (NegGam®, Wintomylon®)

Oxolinic acid (Uroxin®)

Piromidic acid (Panacid®)

Pipemidic acid (Dolcol®)

Rosoxacin (Eradacil®)

Removed from clinical use

Carcinogenic

Carcinogenic

Unavailable

Unavailable

Unavailable

Restricted use

Second generation Ciprofloxacin (Ciprobay®, Cipro®)

Enoxacin (Enroxil®, Penetrex®)

Fleroxacin (Megalone®, Roquinol®)

Lomefloxacin (Maxaquin®)

Nadifloxacin (Acuatim®, Nadoxin®)

Available



Discontinued

Unavailable

8

Norfloxacin (Lexinor®, Noroxin®)

Ofloxacin (Floxin®, Oxaldin®)

Pefloxacin (Peflacine®)

Rufloxacin (Uroflox®)

Restricted use

Discontinued

Unavailable

Unavailable

Table 2.1 Generations of FQs (continued)

Generation Drugs Status

Third generation Balofloxacin (Baloxin®)

Gatifloxacin (Tequin®)

Grepafloxacin (Raxar®)

Levofloxacin (Cravit®, Levaquin®)

Moxifloxacin (Avelox®, Vigamox®)

Pazufloxacin (Pasil®, Pazucross®)

Sparfloxacin (Zagam®)

Temafloxacin (Omniflox®)

Tosufloxacin (Ozex®, Tosacin®)

Unavailable



Available

Restricted use

Unavailable

Restricted use


Unavailable

Fourth generation Clinafloxacin

Gemifloxacin (Fractive®)

Sitafloxacin (Gracevit®)

Trovafloxacin (Trovan®)

Prulifloxacin (Quisnon®)

Unavailable

Available

Unavailable


Unavailable

In development Garenoxacin (geninax®)

Ecinofloxacin

Delafloxacin

Withdrawn due to toxicity issues

9

Table 2.2 FQ antibiotic used in veterinary medicines

FQ antibiotics Brand names Status

Danofloxacin

Difloxacin

ENR

Ibafloxacin

Marbofloxacin

Orbifloxacin

Sarafloxacin

Dicural®, Vetequinon®

Dicural®, Vetequinon®)

Baytril®

Ibaflin®

Marbocyl®, Zenequin®

Orbax®, Victas®

Saraflox®, Sarafin®

Available

Available

Available

Available

Available

Available

Available

2.1.4. Mechanism of action

There are two enzymes, namely DNA gyrase and topoisomerase IV, that have important

roles in DNA replication and proliferation (Blondeau, 2004). DNA gyrase is an essential

enzyme required for bacterial life. Bacterial DNA is generally in equilibrium between a

closed circular double DNA strand conformation and a highly negatively supercoiled

structure. The role of DNA gyrase is to control bacterial DNA topology and chromosome

function by maintaining DNA negative supercoiling. Besides being crucial for DNA

replication and is also responsible for relieving the negative supercoiling, DNA gyrase helps

in bending and folding DNA and removes knots. Topoisomerase IV on the other hand, is

responsible for separating the product of DNA replication, which is the catenated

(interlinked) circular DNA daughter molecule (Higgins et al., 2003).

FQs act by inhibiting DNA gyrase and topoisomerase IV enzymes by irreversibly binding to

the enzyme‐DNA complex and generating a double‐stranded break, resulting in enzyme

denaturation (Hooper, 2001). In terms of FQs’ spectrum activity, these antibacterial agents

show excellent efficacy against Enterobacteriaceae, Pseudomonas aeruginosa, good to

moderate activity against Staphylococci, Mycbacteria, Chlamydia, mycoplasma and

ureaplasma and little or no activity against Streptococci (particularly group D. streptococci),

enterococci and anaerobic bacteria (Brown, 1996).

10

2.1.5. Structure activity relationships of fluoroquinolones

Structure activity relationships (SARs) of FQ antibiotics have been studied intensively to

enhance the efficacy and broaden the antibacterial spectrum activity. An alkyl group at

position 1 (R1) helps in antimicrobial activity. Optimization of the alkyl substitution in the

quinolone structure (i.e. ethyl group in norfloxacin and cyclopropyl group of ciprofloxacin)

has improved antibacterial activity in terms of sensitivity and minimum inhibitor

concentration (MIC) against bacteria. A fluorine atom at position 6 has been shown to

improve the efficacy against Gram negative bacteria and broadens the spectrum activity

against Gram positive bacteria. A basic nitrogen‐containing moiety enhances tissue

penetration and minimises central nervous system toxicity. Furthermore, alteration of

pharmacokinetics of compounds could be done by modifying substitution groups at

positions 2, 5 and 7 of the basic structure (Brown, 1996, Wagman and Wentland, 2007).

2.1.6. Clinical use in animal and human

FQs are synthetic antibacterial agents that are broadly employed for human and veterinary

administration against a variety of bacterial infections (Brown, 1996). There are about 50

FQs used in veterinary and human medicine worldwide. In veterinary medicine, amifloxacin,

ciprofloxacin, danofloxacin, ENR, gatifloxacin, marbofloxacin, norfloxacin are used. The

major FQs used in human medicine include ciprofloxacin, enoxacin, ofloxacin, sparfloxacin,

temafloxacin and tosufloxacin (Brown, 1996). FQs have general pharmacokinetic

characteristics such as good oral absorption (Hooper and Wolfson, 1991), well absorbed

from parenteral injection sites (Gyrd‐Hansen and Nielsen, 1994) and readily distributed to

various tissues in the body (Brown, 1996). The older and modern FQs available in the

market are used in both human and animal medicines, as presented in Tables 2.3 and 2.4.

11

Table 2.3 Older FQs marketed for both human and veterinary uses

Generic name Brand name First introduced Company

Norfloxacin Noroxin 1983 Kyorin/Merck

Pefloxacin Peflacine 1985 Roger Bellon

Ofloxacin Floxin 1985 Daiichi/Ortho

Ciprofloxacin Cipro 1986 Bayer

Enoxacin Penetrex 1986 Dainippon/RPR

Lomafloxacin Maxaquin 1989 Hokuriku/Unimed

Tosufloxacin Tosuxacin 1990 Abbott/Toyama

Temafloxacin* Omniflox 1992 Abbott

Fleroxacin Quinodic 1992 Roche

Nadifloxacin Acutim 1992 Otsuka

Rufloxacin Qari 1992 Mediolanum

*withdrawn from market

Table 2.4 Modern fluoroquinolones marketed for both human and veterinary uses

Generic name Brand name First introduced Company

Levofloxacin Levaquin 1993 Daiichi/Ortho

Sparfloxacin Zagam 1993 Dainippon/RPR

Grepafloxacin Raxar 1997 Otsuka/GW

Trovafloxacin Trovan 1997 Pfizer

Gatifloxacin Tequin 1999 Kyorin/BMS

Moxifloxacin Avelox 1999 Bayer

Gemifloxacin Factive 2003 GeneSoft/LG Life Science

FQs have better potency and activity against most Enterobacteriaceae, fastidious Gram‐

negative species such Haemophillus, including Gram negative cocci, such as Neisseria

gonorrhoeae, Neisseria meningitides, Moraxellacatarrhalis. Ciprofloxacin is the most active

of the available FQs, and inhibits 90% of Enterobacteriaceae at concentrations that are <0.5

12

µg mL‐1. Among all FQs agents, nalidixic acid has the highest minimum inhibition

concentration (MICs) value against these organisms (Hooper and Wolfson, 1991).

In human beings, FQs are highly effective against the majority of bacteria responsible for

urinary tract infection with cure rates of between 90 to 100% in general, with treatment

periods between 7 and 10 days. Norfloxacin, enoxacin and ciprofloxacin can reduce the

symptoms of traveler’s diarrhea in gastrointestinal infections caused by organisms such as

Salmonella, Shigella and phatogenic E. coli and Vibrio species, with a typical treatment

duration being approximately 24 to 90 hours. Ciprofloxacin and ofloxacin have also been

proven to cure skin and soft tissue infections such as cellulitis, superficial wounds and

ischemic ulcers. With regard to lower respiratory tract infections, in which Haemophilus

influenza is the primary pathogen, FQs are better than ampicillin and are comparable or

superior to amoxicillin (Neu, 1992).

For therapeutic uses in animals, only ENR and sarafloxacin are approved in the US; in other

countries such as Indonesia, ENR, norfloxacin and ciprofloxacin are also permitted for use.

ENR is used for complicated and uncomplicated urinary tract infection in dogs with doses

up to 11 mg/kg every 12 hours. ENR is also effective for acute salmonella infections in

calves, colibacillosis in swine, colibacillosis and mycobacterial disease in poultry. Other FQs

such as danofloxacin, has been effective against bovine respiratory disease and

myoplasmosis in poultry (Jordan et al., 1993). Table 2.4 describes the dosages of different

FQs antibiotics for animal medication (Brown, 1996).

13

Table 2.5 Proposed and/or approved dosages of various FQs used in veterinary medicine

Drugs Doses

ENR 2.5 mg/kg every 12 hour, oral for dogs and cats (up to 11 mg/kg for some infections)

5 mg/kg every 24 hour, oral and subcutaneous for pigs

Norfloxacin 22 mg/kg every 12 hour, oral for dogs and cats (deep infections)

Ciprofloxacin 10‐15 mg/kg every 12 hour, oral/slow intravenous for dogs and cats

Danofloxacin 1.25 mg/kg every 24 hour, intramuscular for calves

Flumequine 8 mg/kg/day, oral for one week old calves

15 mg/kg/day, oral for over six weeks old calves

Sarafloxacin 20‐40 ug/mL in drinking water for 5 days (chickens)

30‐50 ug/mL in drinking water for 5 days (turkeys)

2.1.7 Fluoroquinolones used in this study

2.1.7.1. Ciprofloxacin

Ciprofloxacin is a second generation FQ antibacterial that is the most potent against Gram‐

positive bacteria; however, it is less active against Gram‐negative bacteria. It disrupts

bacterial enzymes, DNA gyrase and topoisomerase IV enzymes, in synthesising bacterial

DNA (Wagman and Wentland, 2007).

Ciprofloxacin was first introduced and patented by Bayer A.G. in 1983 before being

approved by the US Food and Drug Administration (FDA) in 1987. Ciprofloxacin is available

as more than three hundred different brand names and is marketed in more than a

hundred countries worldwide. It is formulated into oral dosage forms, intravenous

formulations and external uses (i.e. eye and ear drops). All formulations must be

prescribed by a medical doctor in most countries.

Ciprofloxacin is chemically known as 1‐cyclopropyl‐6‐fluoro‐1,4‐dihydro‐4‐oxo‐7‐(1‐

piperazinyl)‐3 quinoline carboxylic acid, with an empirical formula of C17H18FN3O3 and a

molecular weight of 331.4 g/mol. It is a faintly yellowish to light yellow crystalline

14

substance. Ciprofloxacin hydrochloride (as listed in The U.S. of Pharmacopeia USP, 2007)

exists as the monohydrochloride monohydrate salt of ciprofloxacin. It is a faintly yellowish

to light yellow crystalline substance with a molecular weight of 385.8 g/mol and an

empirical formula of C17H18FN3O3HCl.H2O (Figure 2.3) (Kuijper et al., 2007).

Figure 2.3 Chemical structure of ciprofloxacin.

2.1.7.2. Enrofloxacin

ENR is a synthetic antibacterial agent from the carboxylic acid family of FQs. It has

antibacterial activity against a broad spectrum of Gram‐negative and Gram‐positive

bacteria and is active in both stationary and growth phases of bacterial replication. Its

mechanism of action is not thoroughly understood, however, it is believed to act by

inhibiting bacterial DNA gyrase, thereby preventing DNA supercoiling and DNA synthesis.

The bactericidal activity of ENR occurs within 20‐30 min of exposure.

ENR is chemically known as 3‐quinolinecarboxylic acid, 1‐cycloprpyl‐7‐(4‐ethyl‐1‐

piperazinyl)‐6‐fluoro‐1,4‐dihydro‐4‐oxo, with an empirical formula C9H22FN3O3 of and a

molecular weight of 359.4 g/mol, as shown in Figure 2.4. It is white or slightly yellow

needle‐like crystalline powder, odorless and tasteless and available in hydrochloride,

lactate, and sodium salts. ENR is very soluble in acid or alkaline media, soluble in dimethyl

formamide, slightly soluble in chloroform, methanol and insoluble in water (as listed in the

US of Pharmacopeia USP, 2007). ENR is sold under the trade name Baytril by Bayer

Corporation. It is approved by the U.S. FDA to treat pets and domestic animals. It could be

dissolved in water to treat flocks of poultry; however, due to FQs resistant strains of the

Campylobacter bacteria, it has been withdrawn in September 2005.

15

Figure 2.4 Chemical structure of ENR

2.1.7.3. Norfloxacin

Norfloxacin is a second generation FQs and has a similar mode of action to other FQs drugs.

Norfloxacin was developed by Kyorin Seiyaku K.K from the Japanese Society of

Chemotherapy and patented in 1979. Kyorin later granted Merck and Company, Inc., an

exclusive license in Japan on September 4th 1980. It was then imported in certain countries

including European countries and the U.S. and distributed under the brand name of

Noroxin. Noroxin was approved by the U.S. Food and Drug Administration on October 31,

1986.

Norfloxacin was the first quinolone antibacterial agent with a fluorine atom substituted at

the C‐6 position and a piperazine at C‐7. This substitution enhanced antibacterial activity

compared to the previous quinolone (nalidixic acid) (Carlucci, 1998). Norfloxacin is a 1‐

ethyl‐6‐fluoro‐1,4‐dihydro‐4‐oxo‐7‐(1‐piperazinyl)‐3quinoline carboxylic acid with an

empirical formula of C16H18FN3O3, as shown in Figure 2.5. It is a white to pale yellow

crystalline powder with a molecular weight of 319.34 g/mol and a melting point of about

221°C. It is freely soluble in glacial acetic acid, and very sparingly soluble in ethanol,

methanol and water (as listed in The U.S. of Pharmacopeia, 2007).

Figure 2.5 Chemical structure of norfloxacin.

16

2.1.8. Pharmacokinetics and toxicity

All of the FQs are absorbed through the gastrointestinal tract. The degree of absorption

however, remains variable, from a low absorption rate of 55% for norfloxacin to 95% for

ofloxacin, lomefloxacin, temafloxacin, and pefloxacin. Peak serum concentrations are

achieved 1‐4h after the drug is ingested before a meal, while it reaches maximal peak

serum concentration approximately 2h after a meal. Elderly or patients with poor renal

function will absorb the drug slowly, resulting in delaying peak serum concentration.

Peak serum level after ingestion of 400 mg of norfloxacin and pefloxacin are 1.5 µg mL‐1, 4

µg mL‐1, and 3 µg mL‐1after enoxacin. A 500 mg dose of ciprofloxacin induces a blood level

of approximately 2.5 µg mL‐1 and from 3.5 to 4 µg mL‐1for the 750 mg dose. The half‐lives

of FQs are as follows: norfloxacin 3‐4 h, ciprofloxacin 4 h, ofloxacin 6‐7 h, enoxacin 6 h,

pefloxacin 10 h, lomefloxacin 8 h, temafloxacin 8 h, fleroxacin 8‐10 h, and sparfloxacin 18 h

(Neu, 1992).

Following intravenous administration of FQs such ciprofloxacin, ofloxacin, enoxacin,

fleroxacin, lomefloxacin and pefloxacin, there are no differences in the pharmacokinetics

profile when compare to oral administration. Also, half‐lives of elimination, volume of

distribution, extra renal elimination, and metabolism are the same with oral administration.

FQs, once absorbed into a body, are widely distributed within body tissues and fluids, and

reach high concentrations in many tissues. Interstitial fluid concentrations range from 50 to

100% of the peak serum concentration in the first a few hours, and then it exceeds serum

concentrations. There are different clearance pathways for FQs. Renal mechanism is

important for pefloxacin and lomefloxacin, whereas, hepatic mechanism is important for

pefloxacin. Both renal and hepatic mechanisms are utilised by norfloxacin, ciprofloxacin,

enoxacin, tosufloxacin and sparfloxacin (Neu, 1992).

2.1.7. Adverse effects and drug interactions

FQs are generally well tolerated and the adverse effects are fairly similar among various

quinolone‐based agents. Generally, the overall percentage of adverse reaction occurs in

human is 2 to 4% (Neu, 1992). The most common adverse effect associated with FQs in

human are gastrointestinal (GI) effects such as nausea, vomiting and diarrhea (about 1 to

5%), skin disorder (<2.5%) and central nervous system (CNS) effects, including headaches

17

and dizziness (1‐2%). CNS effects such as sleep disturbances, hallucinations, depression and

seizure are less common. Generally, these adverse effects are mild and self‐limiting

(Mandell and Tillotson, 2002).

Some FQs are highly toxic, such as temafloxacin, grepafloxacin and trovafloxacin. The

‘temafloxacin’ syndrome was characterized by hemolyticanemia, renal impairment,

hepatotoxicity, disseminated intravascular coagulation and hypoglycemia (Mandell and

Tillotson, 2002). Grepafloxacin resulted in serious cardiovascular effects and other severe

adverse effects, such as hepatic eosinophilia and hypoglycemia associated with the use of

trovafloxacin are reported among patients taking those drugs. Due to the severe adverse

effects of those drugs, they are now withdrawn from the market (Mandell and Tillotson,

2002). Table 2.5 shows the incidences of the most common FQs related adverse effects in

human (Neu, 1992).

Table 2.5 Common adverse reactions associated with some FQs

Events (%) Ciprofloxacin Levofloxacin Sparfloxacin Trovafloxacin Lomefloxacin

Nausea

Diarrhea

Taste perversion

Headache

Dizziness

Phototoxicity

5.2

2.3

0.02

1.2

<1.0

0.4

1.2

1.2

0.2

0.1

0.3

<0.1

4.3

4.6

1.4

4.2

2.0

7.9

8.0

2.0

‐

5.0

11.0

<0.03

3.7

1.4

1.0

3.2

2.3

2.4

All FQs interact with multivalent cation‐containing products such as aluminium or

magnesium containing antacids, and products containing calcium, iron or zinc, such as

supplements (Bertino and Fish, 2000). Hence, FQs‐related toxicity can be treated by

administering multivalent cation‐containing products 2 to 4 h after antibiotic ingestion.

Another interaction relates to NSAID‐FQs, in which enoxacin or ciprofloxacin and fenbufen

cause seizures. This interaction occurs because the NSAID of the FQs competitively inhibite

γ‐aminobutyric acid receptors (Hooper and Wolfson, 1991). FQs also interact with

theophylline and other methyl‐xanthines such as caffeine. FQs induce inhibition of the

hepatic CYP‐450 enzyme system, significantly reduce the metabolism of xanthenes,

18

resulting in increasing elimination half‐lives of theophylline and caffeine (Bertino and Fish,

2000, Brown, 1996). This increases the toxicity of xanthenes, especially for patients or

elderly population with renal and hepatic impairment (Wagman and Wentland, 2007).

Moreover, ciprofloxacin can result in nephrotoxicity when it is administered with

cyclosporine (Bertino and Fish, 2000).

2.2. Public health concerns

Many zoonotic foodborne pathogens such as Salmonella, Campylobacter, shiga toxin‐

producing E. coli, Listeria and Yersinia are generally transferred from food animals to

humans (Tollefson and Karp, 2004). FQ antibiotics are currently administered in feed to

treat, prevent, and control infectious diseases and to enhance feed efficiency and

productivity. The use of antibiotics in food‐producing animals could represent a serious

public health concern because it promotes the selection and dissemination of antibiotic

resistant bacteria to humans. The spread of resistant pathogens from food animals to

humans has serious implication to human health, in particular for the treatment of

infection. Many antimicrobial agents including FQs administered to food animals are either

identical or chemically related drugs used in human medicine such as penicillin,

tetracycline and cephalosporins. Once a resistant pathogen occurs, there is a possibility

that genes will encode resistance not just to a particular antibiotic, but to an entire class of

antimicrobials or may cause cross‐resistance to structurally related compounds. This

problem may lead to limitations in treatment choice, resulting in long term medical

complications and high costs, and even treatment failure (Angulo et al., 2000).

The evidence of public health consequences of the use of FQs in food animals has been

documented. Wegener, in 2009, reported that 10 % of 459 Danish patients with

Campylobacter jejuni infections were untreatable with FQs. Study conducted in Minnesota,

US, showed that patients who were infected with resistant C. jejuni and treated with FQs

were found to have a longer duration of diarrhea that lasted almost 3 days. Three extra

days of diarrhea indicated a mild complication of treatment. More importantly,

Campylobacter infections can be serious in vulnerable patients with underlying health

problems. For immune‐compromised patients who have invasive Campylobacter

infections, treatment failure could be fatal (Nelson et al., 2007).

19

2.2.1. Food safety

Veterinary medicines including antimicrobial agents have been widely used to treat

infectious diseases as well as to improve the health of livestock. A variety of drugs such as

antibiotics and feed additives are also being given to animals to artificially enhance weight

gain, improve feed efficiency and productivity. Over the last decade, antibiotic growth

promoters have been intensively administered in animal farming worldwide. In the U.S., for

instance, approximately 80% of the poultry, 75% of the swine, 60% of the beef cattle, and

75% of the dairy calves receive antibiotics at some time in their life.

In terms of human safety, use of FQs in food‐producing animals could lead to presence of

FQs residues in animal derived products. Gips et al., 1994, reported that after

administration of 10 mg norfloxacin/kg body weight at an intramuscular dose to calves,

norfloxacin residue was found in the liver at a concentration of 50‐100 µg kg‐1 after 72‐120

h. Moreover, administering ENR intramuscularly at a dose of 8 mg kg‐1 daily for 4 days led

to both parent drug and oxometabolite persisting in kidney and liver for 12 days at a

concentration of 0.015 µg g‐1 (Anadon et al., 1995).

2.2.2. Maximum residue limits (MRLs)

To protect consumers against unacceptably high residues and to ensure the chemical

safety of food commodities, MRLs must be set by regulatory authorities at national and

international levels. Over the last three decades, two joint FAO/WHO committees have

evaluated a large number of food chemicals, food additives, contaminants, veterinary drug

and pesticide residues. A residue is defined as drug and/or its metabolites still present in

edible tissue of the treated animal at the time of slaughter or that have been passed to

other edible products such as milk and eggs (Heidjen et al., 1999). MRLs are regulatory

tools to specify the maximum amount of a residue of the active ingredients of a particular

veterinary drug that is acceptable in a particular food commodity (Heidjen et al., 1999).

Residue studies provide information about presence and persistence of veterinary drug

residues in edible tissues of the target organs such as muscle tissue, fat, liver and kidney.

Important information reported in residue studies is the method of drug administration (i.e.

in feed or drinking water, per injection or other routes) and the duration of drug

20

administration that should be identical with the intended route of administration and

period of treatment (Heidjen et al., 1999).

With regard to FQ antibiotic residues in animal‐derived food products which might affect

public health, some authorities have established maximum residue limits (MRLs). For

example, the maximum residue limit for ENR in the European Union (EU) is 30 µg kg‐1 for

kidney, liver and muscle in swine, cattle and poultry, which is calculated by summing the

residues of ENR and its major active metabolite, ciprofloxacin (Brown, 1996). In 1998,

European Agency for the Evaluation of Medicinal Products has made a recommendationon

MRL for ENR of 200 µg kg‐1 in ovine kidney and 100 µg kg‐1 in bovine milk (Bucknall et al.,

2003). The MRL values established by the European Union (EU) and the joint FAO/WHO

Expert Committee on Food Additives (JECFA) for FQs and quinolones of veterinary use are

presented in Table 2.6 (Hernandez‐Arteseros et al., 2002).

Table 2.7 MRL values established by the EU and JECFA for residues of FQs and quinolones

used in veterinary medicine(Hernandez‐Arteseros et al., 2002)

Fluoroquinolones Animal species Target tissues MRLs (EU)

(µg kg‐1)

MRLs (JEFCA)

(µg kg‐1)

Danofloxacin Bovine, chicken

Porcine

Muscle

Fat

Liver, Kidney

Milk

Muscle

Skin, fat

Fat

Liver

Kidney

200

100

400

30

100

50

‐

200

200

200

100

400

‐

100

‐

100

50

200

Difloxacin Bovine, porcine

Chicken, turkey

Muscle

Fat, skin, fat

Liver

Kidney

Muscle

400

100

1400

800

300

‐

‐

‐

‐

‐

21

Skin, fat

Liver

Kidney

400

1900

600

‐

‐

‐

ENR and

ciprofloxacin

Bovine, ovine

Porcine,

poultry, rabbit

Muscle, fat

Liver

Kidney

Milk

Muscle, fat

Liver

Kidney

100

300

200

100

100

200

300

‐

‐

‐

‐

‐

‐

‐


used in veterinary medicine (continued) (Hernandez‐Arteseros et al., 2002)


(µg kg‐1)

MRLs (JEFCA)

(µg kg‐1)

Flumequine Bovine, ovine,

Porcine

Chicken, turkey

Salmonidae

Muscle

Skin, fat

Liver

Kidney

Milk

Muscle

Skin, fat

Fat

Liver

Kidney

Muscle + skin in

Natural proportions

200

300

500

1500

50

400

250

‐

800

1000

600

500

1000

1000

3000

‐

500

‐

1000

1000

3000

500

Marbofloxacin Bovine, porcine Muscle, liver, kidney

Fat

Milk

150

50

75

‐

‐

‐

22

Oxolinic acid Bovine, porcine,

Chicken

Fin fish

Muscle

Fat, skin+fat

Liver, kidney

Eggs

Muscle + skin in

Natural proportions

100

50

150

50

300

‐

‐

‐

‐

‐


used in veterinary medicine (continued) (Hernandez‐Arteseros et al., 2002)


(µg kg‐1)

MRLs (JEFCA)

(µg kg‐1)

Sarafloxacin Chicken

Turkey

Salmonidae

Muscle

Skin + fat

Fat

Liver

Kidney

Muscle

Fat

Liver, kidney

Muscle + skin in

Natural proportions

‐

10

‐

100

‐

‐

‐

‐

30

10

‐

20

80

80

10

20

80

‐

2.2.3. Fluoroquinolone resistant bacteria

Resistance to antimicrobial drugs drastically diminish control of many bacterial pathogens.

For highly prevalent foodborne pathogens, such as Salmonella, E. coli and Campylobacter,

the most likely source of resistance is the use of antimicrobial agents in food‐producing

animals (Tollefson, L., 2000). Resistance is usually chromosomally mediated, involving two

enzyme targets of FQs, which are DNA gyrase and topoisomerase IV. DNA gyrase and

topoisomerase IV composed of four subunits (two A and two B) encoded by gyrA and gyrB,

and parC and pare respectively. FQs resistant isolates usually contain one or more

mutations in a small section of gyrA or parC, while gyrB and parE are rare (Piddock, 1998).

23

Bacterial resistance to FQs can also be triggered by a non‐specific mechanism, known as

up‐regulation of drug efflux pumps, and alteration in bacterial membrane that reduces the

drug’s permeation into the cell(Hooper and Wolfson, 1991). Efflux pumps are a primary

mechanism of resistance in Gram negative species such P. aeruginosa in resulting at least

one of the multiple efflux pumps. In contrast, the efflux pumps in Grampositive species

such as P. pneumoniae seems to be limited.

More recently, increased incidence of antibiotic resistance has been reported for

P.aeruginosa, Serratia marcescens and Staphylococci in chronic infections or chronic

bacterial exposure (e.g. inner venous catheter or urinary catheter). FQs resistance to other

antimicrobial agents also may occur, for instance, cross resistance with β‐lactam antibiotics,

aminoglycosides, tetracyclines, macrolide and polypeptide antibiotics, sulfonamides and

diaminopyrimidines (Hooper and Wolfson, 1991).

2.3 Analytical methods for detecting of fluoroquinolones antibiotic residues

There are two main analytical methods to identify and quantify contaminant in foods,

namely instrumental and bioanalytical methods. Instrumental methods include high

performance liquid chromatography (HPLC) with UV/fluorescence detectors (Hung, 2007,

Carlucci, 1998, Espinosa‐Mansilla et al., 2006, Gigosos et al., 2000, Hassonuan et al., 2007a,

Hassonuan et al., 2007b, Holtzapple et al., 2000, Yorke and Froc, 2000, Zheng et al., 2005,

Holtzapple et al., 2001, Holtzapple and Stanker, 1998, Pena et al., 2010, Shim et al., 2003,

Cinquina et al., 2003, Idowu and Peggins, 2004), liquid chromatography mass spectrometry

(LC‐MS) (Marchesini et al., 2007), liquid chromatography tandem mass spectrometry (LC‐

MS/MS) (Bogialli et al., 2008, Ikegawa, 1998, Johnston et al., 2002, Toussaint et al., 2005a,

Toussaint et al., 2005b, Van Vyncht et al., 2002, Dufresne et al., 2007, Volmer et al., 1997),

capillary electrophoresis (Fierens et al., 2000) gas chromatography (GC) (Takatsuki, 1991)

and thin layer chromatography (TLC) and high performance thin layer chromatography

(HPTLC) (Choma et al., 1999, Choma et al., 2004, Choma et al., 2002, Gaugin and Abjean,

1998, Simonovska et al., 1999). Example of bioanalytical techniques is

immunochromatography (Sun et al., 2007, Zhu et al., 2008, Watanabe H. et al., 2002), and

immunosensors(Cao et al., 2007, Giroud et al., 2009, Marchesini et al., 2007, Haasnoot et

al., 2007, Huet et al., 2008, Tsekenis G. et al., 2008). Another immunochemically based

24

method is immunoassays, which rely on specific antigen and antibody interaction. Coupling

of the two methods is high performance immunoaffinity chromatography (HPIAC)

(Holtzapple and Stanker, 1998, Holtzapple et al., 1999, Holtzapple et al., 2000, Holtzapple

et al., 2001, Zhao et al., 2009).

Immunoassays are a method based on antibody‐antigen binding properties, commonly

used to determine low molecular weight contaminants, including veterinary drugs, in food.

Immunoassay plays an important role in ensuring food safety, due to the increasing

number of contaminants in food (Chen et al., 2009a, Chen et al., 2009b). Recently, many

immunoassay as screening tools have been successfully developed as alternatives or

complement to instrument‐based methods for detecting FQs contaminants in food

commodities(Wang et al., 2007a, Bucknall et al., 2003, Burkin, 2008, Li et al., 2008, Huet et

al., 2006, Kato et al., 2008, Tittlemier et al., 2008).

2.3.1. Instrument‐based methods

Instrumental techniques offer high sensitivity and selectivity analyses, and provide highly

accurate and precise results, but this method requires extensive sample preparation,

sample clean‐up which is often time‐consuming, needs large volumes of solvent or

chemical reagents. In addition, they cannot be used with high efficiency in the routine

screening of a large number of sample specimens (Kato et al., 2007), also need highly

trained and experienced individual to operate sophisticated instruments and interpret

complicated results (Beier et al., 1996), and furthermore, it can be very expensive.

2.3.1.1. High performance liquid chromatography (HPLC)

A reversed‐phase high‐performance liquid chromatography has been developed for the

detection of FQs residues in animal derived products in the last two decades. Separation is

usually performed using silica‐based reversed‐phased columns, mainly C18 or C8, but in

some cases phenyl and amides phases have been employed. Due to the residual silanol

groups and metal ions as impurities in column‐packing material, conventional reversed‐

phase columns lead to severe tailing peaks. Therefore, end capped columns or ultra purity

silica columns, such as Inertsil, Kromasil, Puresil, LUNA or Zorbax RX, which are relatively

free of trace metals to strengthen the acidic properties of silanol groups (Hernandez‐

Arteseros et al., 2002) have been encouraged. For acidic quinolone group (AQ), excitation

25

and emission wavelengths are set around 325 and 360 nm respectively, while for

piperazinyl quinolone groups, they are at 275‐280 nm and 440‐450 nm, respectively

(Hernandez‐Arteseros et al., 2002).

Fluorescence detection (FLD) is most commonly employed in HPLC since it is more sensitive

and selective than UV or diode array detection (DAD) (Shim et al., 2003, Hung, 2007, Yorke

and Froc, 2000, Idowu and Peggins, 2004). For instance the limit of detection (LOD) and the

limit of quantification (LOQ) values obtained using FLD were 0.5‐30 µg L‐1and 1µg L‐1,

respectively (Yorke and Froc, 2000, Idowu and Peggins, 2004), which were significantly

lower compared to those established by DAD i.e. 50 µg L‐1and 20 µg L‐1, respectively

(Cinquina et al., 2003). Lower LOD values using FLD were also exhibited, at 25 µg kg‐1 in

chicken tissues (Maraschiello et al., 2001), from 5 to 20 µg kg‐1 in chicken eggs (Zeng et al.,

2005), and LOQ value at 2.4 to 10 µg L‐1 in milk (Marazuela and Moreno‐Bondi, 2004).

2.3.1.2. Liquid chromatography / mass spectrometry (LC‐MS) and (LC‐MS/MS)

Liquid chromatography coupled with mass spectrometry (LC‐MS) is a technique that

merges the physical separation technique of liquid chromatography with the mass analysis

capabilities of mass spectrometry. It is a powerful tool which was developed to overcome

the limitation of HPLC (Toldra and Reig, 2006). This technique has high sensitivity and

specificity, and is used for analysis of compounds that are highly polar, thermally labile and

relatively high molecular weight (Yoon et al., 2003).

Vyncht et al., 2002 determined multi‐residues of FQs in swine kidney with limits of

detection (LOD) that was much lower than the respective residue limits (MRL) by HPLC. LC‐

MS‐MS is another powerful analytical technique that has higher sensitivity and greater

selectivity for determining FQs residues in foods. Toussaint et al., 2005 have demonstrated

the capability of LC‐MS‐MS with LOQ from 1 to 7 µg kg‐1 and LOD from 0.3 to 2.1 µg kg‐1

for11 FQs antibiotics in pig kidney, which were much lower than the MRL values (200 –

1500 µg kg‐1).

2.3.1.3. Gas Chromatography / Mass Spectrometry (GC/MS)

Since FQs are relatively polar and non‐volatile in character, volatile derivatives must be

prepared prior to GC analysis. The most common derivatisation method used is reduction

with NaBH4leading to more volatile properties. Most researchers applied a DB‐5 or

26

equivalent column for the separation with a temperature gradient from 100 to 270oC. In all

cases, detection was carried out by MS in the positive ion mode and signal monitoring is

performed in the SIM mode. GC‐MS is used as a confirmatory tool after determination by

LC‐FLD (liquid chromatography‐fluorescence detector) and also for quantification of

analytes (Hernandez et al., 2002). Takatsuki (1991) developed GC methods to analyse

nalidixic acid, oxolinic acid and piromidic acid in fish and animal edible tissues, resulting in

better sensitivity with the LOD of 1µg kg‐1.

2.3.1.4 Thin Layer Chromatography (TLC) and High Performance Thin Layer

Chromatography (HPTLC)

TLC and HPTLC have been applied successfully for the qualitative and quantitative

detection on multi‐residues in food samples even though their use have rapidly decreased

during the last decade (Toldra and Reig, 2006). These methods are based on

chromatography principle followed by visualization of the separated components bya

means of chromogenic reagent or illuminating with UV light (Toldra and Reig, 2006). Native

fluorescence, indirect fluorescence and terbium sensitized luminescence are other several

detection systems have been used (Hernandez‐Arteseros et al., 2002). The relative

intensity of the spot on the plate can be measured against the internal standard by

scanning densitometry for quantitative analysis, such as detection of clenbuterol and other

agonist drug residues in meat (Toldra and Reig, 2006). The advantages of these methods

are a high throughput, automatisation for higher productivity and that separated sample

can be recovered for further confirmation. However, they have some drawbacks, such as

require skilled and experienced personal, need sample preparation, in some cases, which

can be time consuming, and high false positives.

The above mentioned methods have been used for detecting different FQ residues such as

flumequine in milk samples (Choma et al., 2002), and ciproflaxacin, danofloxacin, ENR,

flumequine, nalidixic acid, norfloxacin and oxolinic acid in pig muscle (Juhel‐Gaugain and

Abjean, 1998) and flumequine and oxolinic acid in edible muscle tissue and fish (Vega et al.,

1995). Gaugain and Abjean (1998) determined seven FQs simultaneously in pork and the

developed method exhibited the IC50 value at 0.96µg kg‐1. Vega et al., 1995 were able to

quantify flumequine and oxolinic acid in edible muscle tissue and fish with a LOD of 0.2 µg

27

kg‐1 and 8‐9 µg kg‐1, respectively. A TLC‐bioautography consisted of a combination of TLC

and microbiological detections directly on the plate has also been developed for detecting

flumequine in milk sample, resulting in an enhanced sensitivity (Choma et al., 2002).

2.3.2. Bioanalytical or immunochemical methods

Food is a complex mixture of lipids, carbohydrates, proteins, vitamins, organic compounds,

and other naturally occurring substances (Van Emon, 2010). It sometimes get exposed to

residues of pesticides, veterinary and human drugs, microbial toxins, preservatives,

contaminants from food processing and packaging, and other residues. These

contaminants can cause difficulties in the analysis of food. Therefore, there is a need for

rapid, simple, and cost‐effective methods for contaminant analysis to secure reliable food

supply. Bioanalytical or immunochemical methods are the method of choice for many food

contaminants (Van Emon, 2010).These methods include immunoaffinity chromatography,

immunosensors, and immunoassays, and are providing important information regarding

the presence of contaminants in food that may impact human health and the environment

(Van Emon et al., 2007).

2.3.2.1. Immunoaffinity chromatography (IAC)

Immunoaffinity chromatography requires sample extraction and cleanup prior to analysis.

The resultant extracts can be coupled with immunochemical assay or instrumentation. Sol‐

gel IAC is a method where the antibodies are trapped in a ceramic SiO2 sol‐gel matrix to

bind target analytes in the samples, and samples are loaded onto a sol‐gel based

immunoaffinity purification (IAP) column. An elution step releases the analytes from the

antibody binding and the analytes are then detected using either an immunoassay or

instrumentation (Van Emon, 2010). The advantages of this technique is utilizing low levels

of organic solvent and small volumes of samples, thus effective in removing interfering

components resulting in high recovery rates with low intereference.

IAC has been applied by many researchers to detect FQ residues in food (Holtzapple et al.,

2001, Sun et al., 2007, Zhao et al., 2009, Watanabe H. et al., 2002). Zhao and coworkers

(2009) successfully developed an IAC method that is coupled to HPLC equipped with a

fluorescence detector for the isolation and purification of 10 FQs in chicken muscle. This

assay provided an analysis with a limit of detection at 0.1 µg kg‐1 for danofloxacin and 0.15

28

µg kg‐1 for other FQs tested. High recoveries from chicken liver muscle, and milk were also

obtained employing this technique, with the mean recovery values being 77 – 96 %, 72 ‐92 %

and 84 ‐ 99%, respectively (Watanabe H. et al., 2002).

2.3.2.2. Biosensors/Immunosensors

Biosensors employ a method composed of a recognition element (e.g. an antibody) and a

transducer that converts binding of an antibody with an antigen into a measureable

physical signal. Such a system is able to detect analyte continuously and selectively,

yielding a response in real time (Van Emon, 2010). Several types of immunosensors have

been used for pesticide and FQ residues detections in foods, including optical, evanescent

wave, surface‐plasmon resonance, fluorescence and electrochemical impedance

spectroscopy (Van Emon, 2010, Cao et al., 2007, Marchesini et al., 2007, Giroud et al.,

2009). This technique provides higher productivity and shorter cycle times which can

analyse multiple residues in one analytical run and up to 120 samples per hour (Toldra and

Reig, 2006). The technique also possesses extremely low detection limit at 1 x 10‐12 g mL‐1

or 3 pmol L‐1, where the signals were detected and characterised by electrochemical

impendance spectroscopy (Giroud et al., 2009). However, the number of biochip arrays

ready to use is still commercially limited. In any case, it requires high operative costs and

initial investment for equipment.

Cao and coworkers (2007) developed a DNA‐based surface plasmon resonance biosensor

for the detection of ENR residue in milk samples. This surface plasmon resonance

biosensor based‐DNA assay works where heating denatured DNA immobilised on the gold‐

coated glass surface. The immobilization was performed by a layer‐by‐layer co‐deposition

with a cationic polymer. The sensor performance was tested with real biological probes.

The detection limit delivered was 3 µg L‐1in milk. Another plasma resonance biosensor

developed for FQ residues able to generate an IC50 values between 2 and 10 µg L‐1 and %

cross‐reactivity between 30 and 100% for five FQs evaluated in chicken muscle (Marchesini

et al., 2007).

2.3.2.3. Immunoassays

Immunoassays are based on the specificity of the antibody and antigen reaction. The

technique has the ability to measure analytes at low concentrations without extensive

29

sample preparation. This method proves to be advantageous for environmental monitoring

where contaminants are typically present at very low levels and theycannot be detected

accurately by other conventional methods without investing on extensive cleanup time.

Hence, this method has been used as a complement to instrumental methods for many

years to detect a wide range of food constituents including substances responsible for

adulterations and from accidental contaminations (Toldra and Reig, 2006).

Specificity electivity is a factor dependent on the complementary nature of an antigen

surface and an antibody binding site. This governs the specificity of an immunoassay. The

affinity constant (Keq), which the ratio of bound and unbound antigen and antibody at

equilibrium as shown in Eq (1), is a measure of how well an antibody can function in an

immunoassay. For food contaminant immunoassay, Keq is typically in the order of 10‐4 to

10‐12 L/mol (Lee and Kennedy, 2007).

Ab + Ag Ab – Ag complex…..(Eq. 1)

Keq (Lmol‐1) = [Ab – Ag] / [Ab][Ag]

Ab = antibody

Ag = antigen

Keq = equilibrium constant or affinity constant

[Ab – Ag] = concentration of bound antigen

[Ab] = concentration of free antibody

[Ag] = concentration of free antigen

An immunoassay provides a fast, simple and cost‐effective method of analysis, with

sensitivity and specificity comparable to or better (in some cases) than the chemical

instrumentation method. One of the key advantages of immunoassay is the speed of

analysis. Immunoassay does not need the laborious sample preparation step, such as

extensive sample clean‐up, to remove interferences. Also, the technique can often handle

numerous samples simultaneously, thereby greatly enhancing sample throughput (Lee and

Kennedy, 2007).

30

2.4. ELISA (Enzyme‐Linked Immunosorbent Assay)

Many immunoassays have been reported for detecting FQs residues and other

contaminants in food matrixes; the most common is the Enzyme Linked‐ImmunSorbent

Assay (ELISA) (Van Emon, 2010). ELISA is a detection system based on enzyme‐labelled

reagents. ELISAs have shown good performance for the analysis of FQ antibiotic residues in

animal meat (Kato et al., 2007, Li et al., 2008, Burkin, 2008, Kato et al., 2008), animal edible

tissues (Duan and Yuan, 2001, Watanabe et al., 2002, Huet et al., 2006, Zhu et al., 2008)

milk (Coillie et al., 2004, Zhao et al., 2007, Kato et al., 2008, Bucknall et al., 2003, Huang et

al., 2010, Duan and Yuan, 2001), eggs (Wang et al., 2007a, Huet et al., 2006), in honey

(Wang et al., 2007a), in shrimp (Tittlemier et al., 2008, Wang et al., 2007a) and eel (Cao et

al., 2011).

2.4.1. Principle

An ELISA involves an enzyme, a protein that catalyses a biochemical reaction and an

antibody or antigen as immunologic molecules. ELISA also requires the stepwise addition

and reaction of reagents to a solid phase bound substance, through incubation and

separation of bound and unbound molecules using washing steps. An enzymatic reaction is

utilised to produce a colour for quantification purposes.

2.4.2 ELISA Format

The key feature of an ELISA is flexibility, as more than one assay format can be formatted

to measure the same analyte. In general, ELISA is divided into three formats; competitive

ELISA, non‐competitive ELISA and double antibody sandwich ELISA. A double antibody

sandwich ELISA is usually employed to measure larger molecular analytes such as proteins,

viruses, and bacteria. A competitive ELISA has been widely employed to detect small

molecular contaminants in food such as mycotoxin, pesticides, and antibiotics. A

competitive ELISA may be either direct (immobilised antibody) or indirect (immobilised

hapten conjugate or antigen) competitive ELISA (Wang et al., 2007a). Whereas, the

principle of a non‐competitive ELISA method is where antigen is bound to the solid phase,

then labelled antibody is then bound to the antigen. The amount of labelled antibody is

then measured. Unlike the competitive format, the results of the non‐competitive assay

are directly proportional to the concentration of the antigen. This is because

31

labelled antibody will not bind if the antigen is not present in the unknown sample (Price

and Newman, 1991).

A direct competitive ELISA refers to an assay in which antibodies are attached to a solid

phase. Then, an analyte and an enzyme‐labelled competing antigen are added together to

compete for the limited antibody binding sites. After incubation, any unbound reagents are

removed by washing. A substrate solution is added to produce a colour for measurement.

As shown on Figure 2.6, an indirect competitive ELISA (based on a solid phase antigen

format) works by which an antigen is attached to a solid phase. After washing of plate, an

analyte and an antibody are added in the test wells. A secondary labelled antibody is used

to detect the antibody that has bound to the solid phase antigen. After an incubation and

washing, a substrate (chromophore solution) is added to develop the colour.

Figure 2.6 Schematic presentation of an indirect competitive ELISA.

The major advantage of a competitive ELISA is the ability to provide specific binding of an

antigen by an antibodyin crude samples. Many matrix constituents co‐present in the crude

sample do not generally affect the antibody’s ability to bind to its antigen (analyte). The

principle of this format is that if more (free) antigens are present in the sample, the fewer

antibodies are available to bind to the antigen immobilised in the well, hence the reference

is termed "competition” (Figure 2.6). Table 2.6 lists ELISA methods developed for the

detection of FQ antibiotics in animal and marine derived products.

32

Table 2.8 ELISAs developed for FQ antibiotics

FQs Antibiotics Limit of detection (LOD)

in µg kg‐1 or µg L‐1

Sensitivity (IC50) in

µg kg‐1 or µg L‐1 Samples References

Sarafloxacin,

difloxacin,

trovafloxacin,

norfloxacin, ENR,

and nalidixic acid

2 7.3 ‐ 48.3 Chicken

liver

(Holtzapple et

al., 1997)

Ciprofloxacin 0.32 n/a* Milk,

chicken,

pork meats

(Duan and

Yuan, 2001)

ENR,

ciprofloxacin,

ofloxacin

0.3 ‐ 0.95 2 ‐ 6 Chicken

kidney, and

milk

(Bucknall et

al., 2003)

ofloxacin,

danofloxacin,

flumequine,

nalidixic acid,

oxolinic acid,

enoxacin and

lomefloxacin

0.21 0.21 ‐ 25 Pig kidney,

muscle,

egg, fish,

and shrimp

(Huet et al.,

2006)

Gatifloxacin 0.05 2.6 Milk (Zhao et al.,

2007)

ENR 0.7 n/a* Milk (Kato et al.,

2008)

Flumequine 12.5 90 Milk (Coillie et al.,

2004)

Ciprofloxacin, 0.095 1.47 Milk (Huang et al.,

2010)

33

Table 2.8 ELISAs developed for FQ antibiotics (continued)




µg kg‐1 or µg L‐1

Samples References

Ciprofloxacin,

ENR,

norlfoxacin,

ofloxacin,

danofloxacin,

pefloxacin,

amifloxacin,

lomefloxacin,

enoxacin,

flumequine,

oxolinic acid,

marbofloxacin,

difloxacin, and

sarafloxacin

0.2 ‐ 3.4 2.1 ‐ 23 Chicken

liver,

muscle,

eggs

shrimp,

honey

(Wang et al.,

2007b)

Norfloxacin,

ENR,

ciprofloxacin,

difloxacin,

sarafloxacin,

ofloxacin,

danofloxacin,

flumequine,

nalidixic acid

and oxolinic acid

0.1 – 17 n/a* Shrimp (Tittlemier et

al., 2008)

34

Table 2.8ELISAs developed for FQ antibiotics (continued)




µg kg‐1 or µg L‐1 Samples References

Norfloxacin,

ENR,

ciprofloxacin,

difloxacin,

sarafloxacin,

ofloxacin,

danofloxacin,

flumequine,

nalidixic acid

and oxolinic acid

0.1 ‐ 17 n/a* Shrimp (Tittlemier et

al., 2008)

Norfloxacin,

ENR,

ciprofloxacin,

difloxacin,

pefloxacin,

cinoxacin,

sarafloxacin,

marbofloxacin,

ofloxacin,

danofloxacin,

flumequine,

nalidixic acid,

oxolinic acid,

enoxacin,

lomefloxacin,

pipemidic acid,

difloxacin, and

orbifloxacin

0.01 0.04 – 10.2 Pork and

chicken

meats

(Li et al.,

2008)

35

Table 2.8ELISAs developed for FQ antibiotics (continued)



Sensitivity (IC50)

in µg kg‐1 or µg L‐1Samples References

Pefloxacin,

fleroxacin, ENR,

ofloxacin,

enoxacin,

norfloxacin,

lomefloxacin,

ciprofloxacin,

sarafloxacin,

gatifloxacin,

pipemidic acid

0.16 6.7 Chicken

liver

(Lu et al.,

2006)

Marbofloxacin

Ofloxacin, ENR,

ciprofloxacin,

and difloxacin

0.6 4.6 Beef and

pork

meats

(Sheng et

al., 2009a)

Danofloxacin,

ENR,

ciprofloxacin,

difloxacin,

norfloxacin,

marbofloxacin,

ofloxacin,

oxolinic acid,

and flumequine

0.1 5.4 Beef,

chicken

and pork

meats

(Sheng et

al., 2009b)

36

Table 2.8 ELISAs developed for FQ antibiotics (continued)



Sensitivity (IC50)

in µg kg‐1 or µg L‐1Samples References

ENR,

ciprofloxacin,

norfloxacin,

ofloxacin,

sarfloxacin,

enoxacin,

levofloxacin,

sparfloxacin,

and pefloxacin

2.5 8.3 Chicken

muscle

tissue

(Chen et

al., 2009a)

2.5. Development of immunoassay for fluoroquinolone antibiotics

2.5.1. Hapten design and synthesis

The initial step in the immunoassay development for FQs is the design and synthesis of

haptens. As FQs have low molecular weight of less than a few thousand daltons, they are

not immunogenic and unable to elicit immune responses in animals. In order to cause

immunogenicity or to produce antibodies against these molecules, they must be coupled

to suitable larger carrier molecules, usually proteins, so that they can stimulate the cellular

immune system. These molecules are called haptens and when coupled to carrier proteins,

they can act as immunogens (Beier et al., 1996), as shown in Figure 2.7 (Hermanson, 1996).

Figure 2.7 An immunogen is made by coupling a hapten to a carrier molecule using a

conjugation reagent.

37

Antigens are usually macromolecules that contain distinct antigenic sites called epitopes

that recognize and interact with the various immune system components (Hermanson,

1996). Antigens are composed of synthetic organic chemicals, lipoproteins, glycoproteins,

RNA, DNA, or polysaccharides as an individual molecule or antigens are parts of cellular

structure such as bacteria, fungi or viruses (Harlow and Lane, 1988).

There are some general criteria in designing haptens for antibody production. The

optimum hapten for a selected target analyte should be a near perfect mimic of the target

structure in size, shape, electronic and hydrogen bonding capabilities and in its

hydrophobic properties (Lee and Kennedy, 2007, Sheng et al., 2009a). An appropriate

functional group for covalent attachment to the protein must be compatible with the

chemistry of the functional group on the target. Moreover, the chemistry of the target

molecule must be understood, and evaluated for methods of insertion of the attachment

handle by established chemistry (Goodrow and Hammock, 1998). Hence, an appropriate

hapten design and synthesis is important in manipulating the specificity and sensitivity of

an antibody (Crowther, 2001, Lee and Kennedy, 2007).

2.5.1.1. Selection of spacer arm point attachment for fluoroquinolone antibiotics

Generally, there are two common selection points of attachment on FQ structure to design

FQ haptens, attached to either a carboxylic group or a piperazynil moiety. Formation of an

active ester of N‐hydroxysuccinimide (NHS) in the presence of carbodiimide is often

employed as a coupling procedure of FQs to the carrier protein. A carboxylate group of FQ

is a target attachment to the carrier protein. This coupling procedure has the advantage

that the active ester acquired is relatively stable under acidic condition, so it is possible to

purify and store the compound. The active ester of NHS reacts quickly with amino acid

groups of proteins, particularly with lysine(Lee and Kennedy, 2007).

A spacer arm of haptens containing 3 to 6 carbons is preferred. A long spacer arm (Lee and

Kennedy 2007, Tittlemier et al., 2008) generally containing more than 6 carbons may lead

to unstable protein or hapten conjugate. Moreover, using bulky functional groups such as

aromatic, cyclic rings or conjugated double bonds could minimise the recognition of this

region by the antibodies (Lee and Kennedy, 2007).

38

From the structural point of view, attaching a secondary amine on the piperazinyl moiety

of FQs to a carrier protein allows the carboxylic acid group to remain unchanged; and this

approach helps to extend the variable moiety away from the point of attachment to the

carrier protein, resulting in good specificity of antibodies (Bucknall et al.,2003; Huet et

al.,2006; Wang et al.,2007; Tittlemieret al.,2008; Li et al., 2008; Burkin, 2008). In addition,

this design exposes 4‐quinolone carboxylic acid common to FQ antibiotics as an

immunodominant region. Consequently, the antibody has high cross‐reactivity of between

10% and 100% (Huet et al., 2006) and between 35‐100% (Wang et al., 2007a) for most

related compounds, making this a useful screening tool.

On the other hand, the carboxylic group on the FQs may be linked to an amino group of the

carrier protein, which enhances the sensitivity and specificity of the antibody. For instance,

Zhao et al., 2007 developed an ELISA test kit using the method which has a limit of

detection of 0.05 µg L‐1 in milk. The results also showed that anti‐gatifloxacin antibody

produced a high specificity towards gatifloxacin. This is due to that gatifloxacin hasa

uniquemethoxy group located in position 8 that may contribute to better specificity.

Coillieet al. (2004) also employed this attachment point to improve specificity of their

antibody. The resulting cross‐reactivity with ENR, ciprofloxacin, difloxacin, danofloxacin,

marbofloxacin and oxolinic acid was less than 0.1%.

In order to obtain better specificity with low cross‐reaction, coupling of a carboxylic acid to

a carrier protein is generally preferred. While a piperazynil moiety coupled to a carrier

protein is more useful when detection of a wide range of FQs is preferred. Apparently,

there is little difference between LOD values of the both approaches. The LOD values of the

carboxylic acid and a piperazynil moeity attachments were 0.32 µg kg‐1 (Duan and Yuan,

2001) and 0.2 µg kg‐1 (Wang et al., 2007a), respectively.

2.5.1.2. Competing hapten to carrier protein ratio

There is a number of proteins used as carriers, e.g. bovine serum albumin (BSA; MW

67,000), ovalbumin (OVA; MW 43,000), keyhole limpet hemocyanin (KLH; MW 4.5 x 105 to

1.3 x 107), aminoethylated (or cationized) BSA (cBSA), human serum albumin (HAS) and co‐

albumin. Generally, BSA and OVA are used as carrier proteinsbecause of their numerous

39

functional groups and high solubility during cross‐linking or even after extensive

modification with hapten molecules (Hermanson, 1996).

DMSO is generally used to solubilize hapten molecules containing bovine serum albumin

(BSA) or ovalbumin (OVA). To maintain the solubility of haptens, a solvent or aqueous

phase mixture may be used in conjugation reactions. BSA remains soluble in the presence

of up to 35% DMSO and precipitates in 45% and above. In contrast, OVA is soluble in up to

70% and precipitates in 80% and above (Hermanson, 1996).

KLH is derived from the mollusk Megathuracrenulata, an extremely large multi‐subunit

protein that contains chelated copper of non‐heme origin. When KLH is dissolved in basic

solution, it produces blue colour solution, while, the solution turns to green when it is

dissolved in an acidic solution. KLH also should not be frozen as the protein denatured

easily, increasing in insolubility, and making conjugation reactions difficult. Stability and

solubility of KLH is preserved in buffers containing 0.9M NaCl, not in buffers containing 0.9%

NaCl. When the concentration of NaCl is less than approximately 0.6 M, the protein will

start to denature and precipitate. Hence, conjugation reaction using KLH should be done

under high‐salt conditions to maintain solubility of the hapten‐carrier complex (Hermanson,

1996).

There are specific requirements for carrier proteins; they must be highly immunogenic and

sufficiently large in size so that they can impart immunogenicity to covalently coupled

haptens. Another criteria for carrier proteins is the presence of suitable functional groups

for conjugation with haptens, maintain high solubility even after extensive modification

with hapten molecules and also present low toxicity in animals’ body (Hermanson, 1996).

2.5.1.3. Conjugation methods

There is a number of conjugation methods employed. They are chosen according to the

functional groups of a hapten, such as hydroxyl, carbonyl, phenol, thiol and sulfhydryl

groups. However, coupling through carboxylic and amine groups remain the most popular

procedure due to the high success rate and high stability of the resulting conjugates(Lee

and Kennedy, 2007).

40

2.5.1.3.1. Carboxylic groups

A carboxylic acid of a hapten is generally conjugated to a protein using either the

anhydride or carbodiimide with N‐hydroxysuccinimide (NHS). Carboxylic groups – mixed

function anhydride is a simple method since it does not require isolation of an active ester.

A carboxylic group of a hapten is converted to an anhydride acid which reacts with an

amino group of a carrier protein in aqueous‐water miscible solvent mixture or reagents

such as isobutylchloroformate (IBCF) with tributyl or triethylamine (Lee and Kennedy,

2007), as shown in Figure 2.8 (Hermanson, 1996).

Figure 2.8 Reaction of a carboxylic acid with a chlorformate forms an amine‐reactive mixed

anhydride.

Carbodiimides are zero‐length cross‐linking agents used to mediate hapten‐carrier

conjugation via carboxylic groups. It is called zero‐length cross‐linking agents, as there is no

additional carbon in forming bonds between the conjugation molecules. N‐substituted

carbodiimides react with carboxylic acids to form highly reactive O‐acylisourea

intermediate which reacts with a primary amine to form an amide bond, soluble isoureais

released as a by‐product (Figure 2.9;Hermanson, 1996).

Figure 2.9 Reaction of a carboxylic acid with carbodiimide forms an O‐acylisourea.

41

There are two basic types of carbodiimides: water soluble carbodiimides, such as 1‐ethyl‐3‐

(3‐dimethyl‐aminopropyl) carbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide

(DCC), 1‐cyclohexyl‐3‐(2‐morphplinyl‐4‐ethyl) carbodiimide methyl p‐toluene sulfonate

(CMC), and water insoluble carbodiimides; such as diisopropylcarbodiimide (DIC). EDC and

DCC are the most common water soluble carbodiimides used for FQ hapten‐carrier protein

conjugation.

Carbodiimide formation effectively occurs between pH 4.5 and 7.5 (Hermanson, 1996).

EDC and CMC are stable in acidic condition (pH 5.5), whereas, DCC is stable in a neutral

environment (pH 7.2). MES (2‐(N‐morpholino)ethanesulfonic acid) or phosphate buffer is

usually used to stabilize the pH during the reactions. In general, the reaction is performed

by derivatizing small molecules with carbodiimides in a water‐miscible organic solvent and

the product is added to an aqueous protein solution for the coupling stage. Organic

solvents may prevent isourea formation in the conugation solution (Wild, 2001).

A carboxylate group conjugated with carbodiimide‐NHS to a protein is often used as a

coupling procedure to develop immunoassays for food contaminants. Many researchers

have employed this coupling procedure to develop better sensitivity and specificity for the

detection FQ residues in seafood and animal‐derived products (Bucknall et al., 2003, Wang

et al., 2007a, Coillie et al., 2004, Duan and Yuan, 2001, Huang et al., 2010).

The active ester obtained from this coupling method is stable under acidic conditions. It is

also highly reactive towards amine functional groups on proteins and other molecules to

form stable amide bonds. Moreover, the conjugation reaction is complete within 2 h at

room temperature and the active ester reacts quickly with the amino groups of proteins

resulting in good yield (i.e., higher epitope density) (Lee and Kennedy, 2007).

The reaction is carried out by activating a carboxylic acid group in an organic solvent with a

mixture of DCC and NHS, and the active ester is added to an aqueous protein solution as

shown in (Figure 2.10;Hermanson, 1996).

42

Figure 2.10 Reaction of a carboxylic acid group with carbodiimide‐NHS forms an active‐

succinimide ester.

2.5.1.3.2. Amine groups

There is a number of ways to conjugate an amine group to a carrier protein. In general,

aromatic amine is converted with nitrous acid to form a diazonium salt. The coupling

reaction is carried out by mixing the diazonium salt and a protein at an alkaline pH. The

reaction mainly occurs with tyrosine, histidine and tryptophan residues of the carrier

protein (Garden and Sporns, 1994). This coupling method produces bonds which are easily

dissociated and has been successfully employed in pesticide immunoassays. For instance,

parathion was converted to a diazonium salt and coupled with bovine serum albumin to

produce specific antibodies (Lee and Kennedy, 2007).

Following the coupling reaction is the conversion of the aliphatic amines with succinic

anhydride carboxylic group to protein. Coupling of aliphatic amines can also be achieved

using 4,4’‐difluoro‐3,3’dinitrophenyl sulfone, cyanuric chloride or toluene 2,4‐diisocyanate.

However, using these reagents attracts some drawbacks; they are hydrophobic, so they

must be dissolved in a solvent before adding to the protein solution, generally resulting in

low conjugation. Hydrolysis may also occur simultaneously, and the active ester of these

43

reagents becomes less available for conjugation. In addition, under alkaline conditions,

these reagents will react with other amino groups containing hydrogen such as sulfhydryl

group of cysteine, phenolic group of tyrosine and imidazole group of histidine (Lee and

Kennedy, 2007).

An alternative approach is available through reacting aromatic amines with succinic

anhydride in pyridine to provide the ester of succinic acid (Wang et al., 1998). Coupling

through succinic acid provides advantages over diazonium salts coupling due to a bridge

group introduced at the same time between hapten and protein, resulting in a more

sensitive assay (Wang et al., 1998). Diazonium salts linking leads to the loss of enzyme

activity as histidine and lysine are present near or in the most active sites of the enzyme

(Lee and Kennedy, 2007). Also, there are no spacer arms between diazonium salts and the

protein, this may result in low assay sensitivity (Wang et al., 1998).

2.5.2. Antibody production

2.5.2.1. Overview

The availability of antibodies with the desired affinity and specificity is the most important

factor governing immunoassay performance. Antibodies are primarily synthesised and

produced by specialized plasma cell lymphocytes (Wild, 1994), its primary function is to

bind antigen specifically or neutralize foreign matters (e.g., on bacterial toxin or viral

penetration of cells) (Deshpande, 1996). A hapten‐protein conjugate (an immunogen) is

used to produce antibody by immunizing an animal (Lee and Kennedy, 2007). There are

generally two techniques for production of antibody, namely polyclonal (via in vivo

technique) and monoclonal antibody (in vitro technique).

2.5.2.2. Polyclonal antibodies

Polyclonal antibodies , sometimes called antisera, are antibodies derived from different B‐

cell lines, a mixture of immunoglobulin molecules secreted against a specific antigen (Beier

et al., 1996). These antibodies are typically produced by immunisation of a

suitable mammal, such as a mouse, rabbit or goat. This induces the B‐lymphocytes to

produce IgG (immunoglobulin G) specific for the antigen. Animals frequently used for

polyclonal antibody production include chickens, goats, guinea pigs, hamsters, horses, mice,

44

rats, and sheep. However, rabbit is the most commonly used laboratory animal due to the

ease of handling (Lee and Kennedy, 2007).

Most of the sensitive immunoassays developed for detection of food contaminants have

been based on polyclonal antibodies (Lee and Kennedy, 2007). Many researchers (Duan

and Yuan, 2001, Bucknall et al., 2003, Coillie et al., 2004, Zhao et al., 2009, Tittlemier et al.,

2008, Huet et al., 2006) applied polyclonal antibodies for detection of FQ antibiotic

residues in edible animal tissues with high sensitivity. For example an immunoassay for

ciprofloxacin (Duan and Yuan, 2001) has a detection limit of 0.32 µg kg‐1. Another sensitive

immunoassay was developed for gatifloxacin (Zhao et al., 2007) has a detection limit of

0.05 µg kg‐1 with an IC50 value of 2.6 µg kg‐1.

There is no standardised protocol for immunization. Injection of the immunogen is done

via two steps, beginning with an initial injection and then followed by subsequent boosting

injections according to a regular schedule (Lee and Kennedy, 2007). Immunogens are

prepared by emulsifying hapten conjugated protein with 0.9% NaCl (saline) and an

adjuvant. The optimal initial boosting dose for rabbit is about 100 µg per mL, whereas for

subsequent boosting a much lower immunogen level is required; about 5‐20 µg per mL

(Wild, 1994). In terms of blood collections, the first bleed is taken about 14 days after the

booster immunization, while subsequent bleeds are taken at regular intervals, usually

monthly. The main routes of immunization for polyclonal antibody production for rabbit

are generally either subcutaneous, intradermal or intramuscular routes or a combination of

these (Lee and Kennedy, 2007).

2.5.2.3. Monoclonal antibodies

Monoclonal antibodies are those produced by one type of immune‐cell clones originated

from a single parent cell, called hybridoma cell. Mice are the most commonly animal used

to produce monoclonal antibodies. Hybridomas are cell lines produced by fusion of an

immunized B‐lymphocyte and myeloma (tumor) cell (Lee and Kennedy, 2007). Since an

individual lymphocyte produces only a single antibody type, all of the antibody molecules

produced by a hybridoma cell line are identical. Thus all of the antibodies have the same

amino acid sequence and binding properties. In contrast, polyclonal antibodies are

produced by immunizing with a hapten protein conjugate, resulting in a mixture of

45

antibodies with different binding properties. For example, antibodies produced by binding

specifically with the hapten‐linkage chemistry complex will have different affinities and

cross‐reactivities (Beier et al., 1996).

Some reseachers have reported sensitive immunoassays based on monoclonal antibodies.

For example, immunoassay for ENR (Kato et al., 2007) exhibited a detection limit of 0.76 µg

kg‐1. Another sensitive immunoassay using monoclonal antibody was developed for

norfloxacin (Li et al., 2008) and had a detection limit of 0.01‐1 µg kg‐1 which was

comparable to other immunoassay based on polyclonal antibodies. Some monoclonal

antibodies displayed lower sensitivity and higher limit of detection values compared with

polyclonal antibodies. Bucknall et al. (1997) used monoclonal antibodies to develop

sarafloxacin, difloxacin, troflavoxacin, norfloxacin, ENR and nalidixic acid and reported a

limit of detection of 10 to 100 µg kg‐1. Monoclonal antibodies were also used by Zhu and

coworkers (2008) to develop assays for norfloxacin and perfloxacin. The assay showed the

sensitivity at 25 µg kg‐1 for the targeted FQs and for other FQs (ENR, ciprofloxacin,

flumequine, ofloxacin, lomefloxacin, enoxacin, danoxacin and marbofloxacin,) the

sensitivity was at 100 µg kg‐1.

2.5.3. Immunoassay format

There are two classifications of immunoassays; the homogeneous immunoassay where the

detection system is measured without separation of free and antibody bound components,

and heterogeneous immunoassay where the measurement is conducted after a separation

of free and antibody bound components (Lee and Kennedy, 2007). The heterogeneous

immunoassays are often employed to generate sensitive immunoassays by utilizing an

enzyme as a detection system. The sensitivity of immunoassay determined by antibody

affinity, type of label or detection system used for the label, type of format employed and

manipulation of reagents in a given format (Deshpande, 1996).

Heterogeneous immunoassays can be formatted as either competitive or non‐competitive.

In a competitive immunoassay format, free analytes compete with labelled antigens for

limited antibody binding sites. The amount of labelled antigen bound to antibody site is

inversely proportional to antigen concentration. In non‐competitive immunoassays

format,in contrast to a competitive format, antibody‐antigen binding occurs in excess of

46

antibody (labelled). The amount of labelled antibody measured is directly proportional to

antigen concentration (Crowther, 2001).

2.5.4. Assay characterization

In order to determine the efficacy of an ELISA test kit for detecting FQs in animal‐derived

products, it is important to fully characterise a competitive immunoassay for its analytical

parameters. Antibody characterization includes the determination of sensitivity, limit of

detection, specificity, matrix interferences, data precision and accuracy and reagent

stability. In competitive immunoassays, presence of analyte is reflected as inhibition of

colour development with a log‐linear relationship between analyte concentration and

colour development. Thus, the curve is typically sigmoidal in shape, when plotted

absorbance vs concentration in a log‐scale format (Lee and Kennedy, 2007).

The % control absorbance is determined by Eq. (2) where it is determined by adding a

sample matrix without FQs with enzyme conjugate as maximum colour generated by the

assay. The blank is usually generated for determination of background (Lee and Kennedy,

2007).

2..........100/% Eq

AA

AABoB

blankcontrol

blank

A = absorbance

Acontrol = absorbance of analyte at zero concentration

Ablank = absorbance of blank wells

2.5.4.1. Calibration curve

The calibration curves (dose‐response) of the analytes in the immunoassay are usually

obtained by plotting concentration on the X‐axis against either absorbance (A) or %

inhibition on the Y‐axis.

2.5.4.2. Sensitivity, limit of detection (LOD) and limit of quantification (LOQ)

Sensitivity is the competence of an assay to detect the smallest amount of a target analyte

under defined conditions (Crowther, 2001). Sensitivity of animmunoassay is generally

determined by an IC50 value, which is the concentration of an analyte required to reduce

47

the colour development by 50%. Equation 3 shows the determination of %Inhibition for

each standard:

3..........1001% Eq

AA

AAInhibition

blankcontrol

blank

Where:

A = average absorbance for each standard and sample

Ablank = average absorbance for blank

Acontrol = average absorbance for control

Blank = matrix with no analyte, containing only solvents, with enzyme

conjugate

Control = matrix with no analyte, containing solvents and diluents, with

enzyme conjugate

A limit of detection is determined by the concentration of analyte that reproducibly

provides either 15% or 20% inhibition of colour development (IC15 or IC20) which must lie

within the linear portion of the curve(Lee and Kennedy, 2007).

Sensitive immunoassays have been described for FQ antibiotics, with the limit of detection

ranging from 0.05 µg kg‐1 to 50 µg kg‐1. The required assay sensitivity and limit of detection

vary depending on intended use or the legal requirement. For example, the maximum

residue limits (MRLs) for various FQs in meat products, poultry, fish and milk is established

by the European Union (CR ECC, 1990) from 10 to 1900 µg kg‐1. However, some

immunoassays are designed to detect lower concentrations, for example, in environmental

water to meet legal requirements at very low part‐per‐billion or high part‐per‐trillion(Lee

and Kennedy, 2007).

2.5.4.3. Specificity and cross reactivity

Specificity of the immunoassay can be defined as the ability of the assay to detect the

analyte of interest in a heterogeneous mixture (Wang et al., 1999). The ability depends on

the properties of the antibodies as well as the properties of each set of immuno‐reagents

48

used for the assay. Generally, better specificity can be obtained by employing monoclonal

antibodies than polyclonal antibodies since they react as a single population of antibody

with a single affinity (Crowther, 2001).

Cross‐reactivity is defined as the ability of a population of antibodies to bind different

molecules other than the analyte(s) of interest. Hence, cross‐reactivity is a measure of

antibody responses to substances other than the analyte of interest and is directly related

to the specificity of immunoassay. The specificity can be determined by cross‐reactivity of

an antibody using compounds of structural similarity. The cross‐reactivity of FQs antibiotics

is calculated using the following formula:

%CrossReactivity IC50ofENR

IC50ofotherfluoroquinolonesx100… . . . 4

2.5.4.4. Matrix interference

One key advantage of immunoassay is that it does not need extensive sample preparation.

Hence, immunoassay is generally less affected by sample matrix than the instrument‐based

methods. Generally, water samples are analysed as is or diluted form. For example, milk

samples are diluted with purified water or buffer, and animal food or edible tissues derived

from animal sources are extracted with a water miscible solvent and the extract is then

analysed after simple dilution with purified water or buffer.

It is known that various substances present in complex biological system can affect

antigen‐antibody interaction in an immunoassay and can reduce the sensitivity and

reliability of the immunoassay. There are a number of ways to reduce matrix effects;

perform either sample interfering removal procedure, sample extraction or dilute the

sample solution. Zhao et al., (2007) employed the latter method to maintain the sensitivity

of the assay, by diluting defatted milk with PBS buffer.

The matrix effects can be determined by comparing the standard curve of the analyte‐free

matrix and the standard curve prepared in certain pH buffers or solvent system (Wang et

al., 1998; Lee and Kennedy, 2007) or by contracting dose‐response curves with the real

sample spiked, including a blank (Henniona and Barcelo, 1998; Lee and Kennedy, 2007). If

these two curves are superimposed on one another, it means that the matrix effect is

49

insignificant. However if the curves shift either to left or right, it corresponds to a loss or

increase in the sensitivity of the assay.

2.5.4.5. Assay accuracy and precision

The accuracy of an immunoassay is generally determined by two ways. First, immunoassay

data are compared to the levels of analyte spiked into a food matrix. This is usually used

when the performance of the immunoassay is initially validated. Second, by comparing the

results obtained in immunoassay data with those obtained from instrument‐based analysis

methods such HPLC, GC or MS. There are three parameters to correlate between

immunoassay data and instrument‐based analysis; these include: 1) determining the

correlation coefficient as either r or r2; the value should usually be greater than 0.99 ±

0.001, 2) the slope of the regression line should be 1, and 3) how close the plot is from the

origin and generally the regression plot should pass above the origin (Lee and Kennedy,

2007).

2.6 Conclusion

Infectious diseases are now becoming a major problem for the livestock industries and may

affect the productivity, which may result in a decrease of food‐producing animals. Hence, a

wide variety of antibacterial drugs is administered to prevent and treat diesease. FQs are

one family of synthetic antibiotics with broad spectrum antibacterial activity, which has an

important application in veterinary medicine for the treatment of antibacterial infections.

FQ antibiotics have been intensively used in animal treatment. This has led to the presence

and accumulation of FQ residues in food, posing potential risks in the development of FQs

resistant bacteria in animals. This may have an implication to human health, in particular

for the treatment of human infection.

Maximum residue limits (MRLs) are one food safety parameter usedto ensure the

antibiotic safety and protect consumers from unacceptably high residue levels in food

commodities. The maximum residue limits (MRLs) of FQs in seafood and animal‐derived

products have been established by various authorities such as the European Union (EU),

the joint FAO/WHO Expert Committee on Food Additives (JECFA). These authorities have

established MRLs values for FQs and their metabolite between 100 and 300 µg L‐1 in

muscle tissues, fat and milk for all species (Hernandez‐Arteseros et al., 2002).

50

There are two common analytical methods applied to monitoring and determine FQ

residues in foodstuffs of animal origin. These are instrument‐based methods (i.e. HPLC,

LC/MS, LC‐MS/MS GC/MS, and HPTLC) and bioanalytical or immunochemical methods

(immunoaffinity chromatography, immunosensors and immunoassays). Instrumentation

methods suffer from drawbacks such as extensive samples preparation which is time

consuming and laborious. They also need trained personnel to run instruments properly,

require high operating costs and require very expensive equipment. An immunochemical

assay, such as immunoassay is an alternative method that complements instrumental

methods. The advantages of immunoassays are low operating costs, minimise reliance for

highly trained staff, and moreover, it is convenient and rapid performance for screening

purposes.

51

CHAPTER 3. HAPTEN SYNTHESIS

3.1 Introduction

The key step in developing the immunoassay for fluoroquinolone (FQ) antibiotics is the

design and synthesis of optimum haptens (Figure 3.1). Several general criteria have been

established in designing ideal haptens for antibody production. For a selected target

analyte, an optimum hapten should be a near perfect mimic of the target structure in size,

shape, electronic and hydrogen bonding capabilities as well as hydrophobic properties

(Skerritt et al., 1995, Goodrow et al., 1995). Another criterion is the selection of functional

groups for a covalent attachment to a carrier protein, which must be compatible with the

chemistry of the functional groups on the targets (Goodrow and Hammock, 1998b). In

general, a spacer arm length of three to six carbons is preferable, and the use of the bulky

functional groups such as aromatic, cyclic rings or conjugated double bonds should be

avoided to minimise the recognition of this region by the antibodies (Lee and Kennedy,

2007).

Figure 3.1 FQ drugs used in synthesising haptens

There are two common selection points for the attachment of linkers onto the FQ

structures, namely the carboxylic acid group or piperazynil group (Figure 3.2). Treatment of

the carboxyl group with N‐hydroxysuccinimide (NHS) in the presence of carbodiimide as a

coupling reagent has often been used to link FQs to carrier proteins via a peptide bond.

This gives advantages such as the resulting active ester is stable under acidic conditions, so

it is possible to purify and store the compound. An active ester of NHS reacts quickly with

amino groups of proteins resulting in good epitope density (Lee and Kennedy, 2007).

Authors who have synthesised FQs by using the carboxylic acid moiety as a point of linker

attachment, have reported highly specific antibody with cross‐reactivity of less than 0.1%

52

for danofloxacin and flumequine, respectively (Sheng et al., 2009b, Yu et al., 2010, Coillie et

al., 2004). This hapten design is useful to develop a specific single FQ assay which is called a

specific assay.

Figure 3.2 Point of the attachment of linkers on FQ antibiotics

Others believed by designing FQ haptens using the secondary amine group located in the

piperazinyl moiety as the point of attachment would result in antibodies that are highly

cross‐reactive to other FQs. The cross‐reactivity of antibodies obtained by employing this

approach was between 10 and 100% for most structurally related FQ compounds (Huet et

al., 2006, Wang et al., 2007a, Burkin, 2008). Such assays are only useful as a screening tool

to detect several FQ antibiotics simultaneously (this is termed a generic assay).

With regard to conjugating a hapten either directly or indirectly to a carrier protein to

produce a specific or generic assay, there are many studies have been reported. ENR

haptens were synthesised by directly conjugating the analyte to a carrier protein (Wang et

al., 2007b, Bucknall et al., 2003, Kato et al., 2007, Cao et al., 2009), which resulted in ELISAs

with high cross‐reactivity to other FQ drugs and generated cross reactive assays. In this

study, indirect conjugating hapten to a carrier protein was introduced to develop a specific

assay for a specific single compound of FQs.

Several antibodies and immunoassays have been developed for ciprofloxacin (Burkin, 2008,

Bucknall et al., 2003, Wang et al., 2007a, Duan and Yuan, 2001) and norfloxacin (Wang et

al., 2007a, Huet et al., 2006) based on direct conjugation of the targets to carrier proteins.

The generated assays exhibited broad specificity for the FQs and were capable of detecting

multiple targets simultaneously.

Tittlemier et al. (2008) developed a generic ELISA by introducing a 6‐bromohexanoic acid

linker to the piperazynil group of norfloxacin. The resulting assay had little moderate cross‐

53

reactivity, i.e., detected 4 out of 11 FQs tested and had limit detection values from 1 to 17

µg L‐1 for the four FQs. The criticism about this approach is that this hapten design formed

another carboxylic acid group on the spacer arm, resulting in two carboxyl groups in space

before the conjugation to a carrier protein. Both of these carboxyl groups could attach to

the carrier protein, potentially forming a cyclic structure on the protein surface. As well,

long spacer arm leads to unstable hapten and protein conjugates (Li et al., 2008).

Meanwhile, Li et al., (2008) introduced a suitable spacer arm in length(two carbons) for

synthesising a norfloxacin hapten, which resulted in a sensitive assay with an IC50 value at

0.6 µg L‐1 and high cross‐reactivity between 10 and 100% for 13 out of 17 FQs tested.

In this study, we approached conjugation of FQ haptens to carrier proteins by designing

novel ENR, ciprofloxacin and norfloxacin haptens. We employed both piperazinyl and

carboxyl moieties of the FQs for spacer arm attachment but took different synthesis routes

to compare the cross reactivity and specificity of different conjugates. The carboxyl group

of ENR was attached to a four carbon linker of tert‐butyl β‐alanine, to generate a highly

specific assay. Conversely, the piperazinyl moiety of ciprofloxacin and norfloxacin was

linked with 1,4‐dibromobutane and 4‐bromocrotonic acid, respectively to generate assay

with broader specificity. Preparation of conjugates of derivatives ENR, ciprofloxacin and

norfloxacin haptens to carrier proteins was carried out by employing a NHS active ester

coupling method.

3.2 Materials and instrumentation

3.2.1 Materials and chemicals

3.2.1.1 Materials

TLC plates were purchased from Merck Chemical Ltd., silica gel H (TLC grade), neutral

alumina and celite were bought from Sigma Aldrich, USA. Silica gel H was obtained from

Merck, Darmstadt, Germany and solvents for NMR such as dimethylsulfoxide (DMSO‐d6),

deuteriochloroform (CD3Cl) and deuterium oxide (D2O) were obtained from Cambridge

Isotope Laboratories, Inc. USA.

3.2.1.2 Chemicals

ENR, ciprofloxacin, norfloxacin, methyl 4‐bromocrotonate, dicyclohexylcarbodiimide (DCC),

N‐hydroxysuccinimide (NHS), tert‐butyl β‐alanine, 4‐di‐(methylamino) pyridine (DMAP)

54

were bought from Sigma Aldrich, USA. ENR HCl, ciprofloxacin HCl and norfloxacin HCl were

obtained from Ruland Chemistry Company Ltd., China. 1.4‐dibromobutanewas purchased

from Aldrich Chemical Company, inc. 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide

hydrochloride (EDC) was purchased from Alfa‐Aesar.

3.2.2 Equipment and instrumentation

3.2.2.1 Thin Layer Chromatography (TLC)

The Rf values refer to TLC on alumina or silica gel 60 F254 pre‐coated plates with

visualisation under UV light.

3.2.2.2 Nuclear Magnetic Resonance (NMR) spectroscopy

Proton (1H), carbon (13C) and 135DEPT‐NMR were recorded on Bruker DPX 300 Instrument

(300 MHz). Chemical shifts were reported in δ (ppm), coupling constants in J (hertz) and

the following abbreviations were used to describe multiplicities: s, singlet; t, triplet; q:

quartet; dd, doublet of doublet; dt, doublet of triplet; m, multiplet. DEPT stands for

Distortion less Enhancement by Polarization Transfer, and was used to determine the

presence of primary, secondary and tertiary carbon atoms. The DEPT method differentiates

between CH, CH2 and CH3 groups by variation of the selection angle parameter (45 o, 90 o

and 135o). 135DEPT gives all CH and CH3 in a phase opposite to CH2.

3.2.2.3 Mass Spectrometry

All electrospray ionization (ESI) mass spectrometry (MS) spectra were carried out using an

Agilent MD‐1100 ESI/APCI LC‐MS (Bioanalytical Mass Spectrometry Facility, the University

of New South Wales (UNSW)) and using Bruker‐FTMS 4.7T LC‐MS/MS (Monash University).

The following abbreviations were used for MS: HRMS; high resolution mass spectrometry,

LRMS; low resolution mass spectrometry.

3.3 Hapten Synthesis

There are two main synthetic routes of spacer arm attachment to FQ hapten employed in

this study. These protocols are described as follows:

55

3.3.1 The attachment via carboxylic group of FQs as a spacer arm for ENR acid hapten

ENR was first attached to a linker of tert‐butyl β‐alanine via the carboxylic group of ENR to

give the compound 1 (scheme 1) which was treated with trifluoroacetic acid (TFA) to yield

acid2 (scheme 2) without further purification.

3.3.1.1 Synthesis of tert‐butyl 3‐(1‐cyclopropyl‐7‐(4‐ethylpiperazin‐1‐yl)‐6‐fluoro‐4‐oxo‐

1,4‐dihydroquinoline‐3‐carboxamido)propanoate, [ENRtert‐butyl], compound (1),

scheme 1

ENR (500 mg, 1.39 mmol) was dissolved in dry dichloromethane, DCM (20 mL) and cooled

in an icebath for 15 min. Dicyclohexylcarbodiimide, (DCC, 574 mg, 2.78 mmol) and

dimethylaminopyridine, (DMAP, 20 mg) were added and the mixture was stirred for a

further 15 min. Tert‐butyl β‐alanine (404 mg, 2.78 mmol) was then added and the mixture

was stirred overnight at room temperature. A white precipitate of dicyclohexylurea by‐

product was removed by filtration through celite. The crude reaction mixture (1.5 g) was

purified by gravity column (i.e. packed with neutral alumina; column size; diameter: 30 mm,

length: 20 cm; flow rate: 5 mL/min) using ethyl acetate as an elution solvent and the

fraction was evaporated to dryness under vacuum, giving a white compound (808 mg, 55%).

The purified product (compound 1) was then confirmed by TLC (Al2O3), Rf = 0.45 (ethyl

acetate). 1H‐NMR (300 MHz, DMSO‐d6): δ 1.43‐1.58 (m, 3H, [CH3], 1.43‐1.58(m, 4H,

[N(CH2)2]‐1) 1.86 (s, 9H, [3CH3], tert‐butyl group), 2.84‐2.95 (m, 2H, [CH2]‐5’), 3.02 (s, 4H,

[CH2]‐2’3’), 3.60‐3.67 (m, 4H, [N(CH2)2]‐1’,4’), 3.95 (q, J = 6.0 Hz, 2H [CH2]‐4’), 4.18 (t, J = 4.5

Hz, 1H, [N(CH)]‐1), 4.48 (q, J = 7.5 Hz, 2H [CH2]‐3”), 7.03 (q, J = 3.0 Hz, 1H, [CH]‐8), 7.93 (d, J

= 6.0 Hz, 1H [NH]‐2”)., 8.29 (d, J = 6.0 Hz, 1H, [CH]‐5), 8.54 (s, 1H, [CH]‐2).13C‐NMR (75 MHz,

DMSO‐d6):δ 7.89 (C‐1), 12.29 (C‐6’), 29.07 (C‐4”), 30.70 (N‐C‐1), 33.71 (C‐7”), 47.86 (C‐1’,4’),

49.82 (C‐2’,3’), 51.90 (C‐5’), 80.37 (O‐C‐6”), 107.80 (C‐5), 110.33 (C‐8), 139.31 (C‐3), 146.98

(C‐2), 149.17 (C‐6), 154.41 (C‐7), 156.96 (C‐1”), 167.20 (C‐5”), 171.11 (C‐4).


dihydroquinoline‐3‐carboxamido)propanoic acid, [ENR acid], compound (2), scheme 2

Compound 1 (808 mg, 1.66 mmol) was treated with trifluoroacetic acid (TFA) 1 mL at room

temperature for 30 min. The excess TFA was removed under vacuum to yield a crude acid

as an oily yellow product with a yield of 395 mg (49%) which was used in the next step

without further purification. The cleavage of the tert‐butyl group (compound 2) was

confirmed by TLC (Al2O3), Rf = 0.13 (ethyl acetate).1H‐NMR (300 Hz, DMSO‐d6): δ 0.60‐0.09

56

(m, 3H [CH3], 0.60‐0.09 (m, 4H [N(CH2)2]‐1) 1.82‐1.86 (m, 2H [CH2]‐5’), 2.60‐2.72 (m, 4H

[CH2]‐2’3’), 2.78‐2.90 (m, 4H, [N(CH2)2]‐1’,4’), 2.99 (s, 2H [CH2]‐4’’), 3.10 (t, J = 3 Hz, 1H,

[N(CH)]‐1), 3.19‐3.36 (m, 2H [CH2]‐3”), 6.38 (d, J = 9 Hz, 1H, [CH]‐8), 6.99 (d, J = 9 Hz, 1H

[NH]‐2”)., 7.26 (d, J = 12 Hz, 1H, [CH]‐5), 8.00 (s, 1H, [CH]‐2), 9.33 (t, J = 6, 1H [COOH]‐

6”).13C‐NMR (75 Hz, DMSO‐d6): δ 7.92 (C‐1), 9.28 (C‐6’), 30.07 (C‐4”), 31.85 (C‐3”), 33.68

(N‐C‐1), 47.89 (C‐1’,4’), 50.46 (C‐2’,3’), 55.73 (C‐5’), 107.20 (d, J = 15.75 Hz, C‐8), 110.49 (C‐

3), 138.66 (C‐5), 139.47 (C‐9), 147.32 (C‐10), 149.92 (C‐2), 156.99 (C‐7), 158.47 (C‐6),

158.96 (C‐1”), 164.23 (C‐5”), 173.42 (C‐4). ESI‐LRMS: calculated for C22H27FN4O4: 430.20,

found m/z 431.30 [M + Na]+; and for relative abundance (%): 430.20 (100.0%), 431.20

(25.3%), 432.21 (3.6%), found m/z 431.30 (100.0%), 432.30 (26.5%), 433.30 (3.5%).

Scheme 1. Synthesis of compound 1 (denoted as tert‐butyl ENR hapten)

NN

N

F

H

O O

+

OH

H2N

O

O

DCM, DCC, DMAP

NN

N

F

H

O O

NH

O

O

H3C

H3C

Enrofloxacin

tert-butyl beta alanine

tert-butyl 3-(1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxamido)propanoate(enrofloxacin tert-butyl)

RT, 24 hrs

57

Scheme 2. Synthesis of compound 2 (denoted as ENR acid hapten)

3.3.2 The spacer arm attachment via the piperazinyl moiety of ciprofloxacin to create

ciprofloxacinbutyl NHS ester hapten

The ciprofloxacin butyl NHS ester hapten was derived via the conjugation of 4‐

bromobutane NHS ester linker to the piperazynil group on ciprofloxacin. 1,4‐

Dibromobutane was attached to NHS under basic condition (pH 9.5) to give a linker of 4‐

bromobutane NHS (1‐[4‐bromobutoxy]pyrrolidine‐2,5‐dione), compound 3 (scheme 3).

Compound 3 was then coupled via the piperazinyl group of ciprofloxacin to form

compound 4 (scheme 4). The product was purified using flash column chromatography

packed with silica gel (column size; diameter: 30 mm, length: 20 cm; flow rate: 1 mL/min;

eluent: ethyl acetate/MeOH = 8:2).

3.3.2.1 Synthesis of 4‐bromobutane NHS ester linker, compound (3), scheme 3

1,4‐Dibromobutane (1032 mg, 4.78 mmol) was added to N‐Hydroxysuccinimide (NHS; 500

mg, 4.34 mmol) dissolved in dried dimethylsulfoxide (DMSO; 10 mL),=The mixture was

stirred for 15 min. Anhydrous potassium carbonate(K2CO3; 500 mg) was added to the

reaction mixture and stirred for 36 h at room temperature (approximately at 25oC). The

crude reaction mixture was washed with saturated brine solution (3 x 15 mL) and extracted

with ethyl acetate (30 mL). The aqueous layer was removed and ethyl acetate layer was

filtered through anhydrous sodium sulphate. The organic layer (ethyl acetate) was

58

evaporated to dryness under vacuum, yielding a brown viscous product (700mg, 35%).

Compound 3 was then confirmed by TLC, Rf = 0.67 (ethyl acetate) 1H‐NMR (300 Hz, CDCl3):

δ 1.88‐1.92 (m, 2H, CH2), 2.06‐2.11 (m, 2H, CH2), 2.70 (s, 4H, 2CH2, cyclopentana), 3.50 (t, J

= 6 Hz, 2H, CH2‐Br), 4.11 (t, J = 6 Hz, 2H, O‐CH2).

3.3.2.2 Synthesis of 1‐cyclopropyl‐7‐(4‐(4‐((2,5‐dioxopyrrolidin‐1‐yl)oxy)butyl)piperazin‐

1‐yl)‐6‐fluoro‐4‐oxo‐1,4‐dihydroquinoline‐3‐carboxylic acid, [ciprofloxacin butyl NHS

ester hapten], compound (4), scheme 4

Ciprofloxacin HCl (250 mg, 0.78 mmol) was dissolved in dried dimethysulfoxide (5 mL), and

compound 3 (375 mg, 1.55 mmol) was added and stirred for 30 min. Sodium hydride,

(NaH,45 mg, 1.88 mol) was added and the mixture was refluxed in a flask immersed an oil

bath at 60‐70oC for 36 h. The reaction mixture was washed with petroleum ether (3 x 10

mL) and extracted with DCM (10 mL). The organic layer (DCM) was evaporated to dryness

under vacuum. The crude product was purified by the chromatography column(i.e. packed

with silica gel; column size; diameter: 30 mm, length: 20 cm; flow rate: 1 mL/min; eluent:

ethyl acetate/MeOH = 8:2) and evaporated to dryness under high vacuum, giving a pale

brown product (430 mg, 65%).Compound 4 was then confirmed by TLC, Rf = 0.26 (ethyl

acetate/MeOH = 8:2) 1H‐NMR (300 Hz, DMSO‐d6): δ 1.52‐1.60 (m, 4H, [N(2CH2)]‐1) 2.31‐

2.44 (m, 2H, [CH2]‐6’), 2.31‐2.44 (m, 2H, [CH2]‐7’), 2.67 (s, 4H, [2(CH2)]‐3”4”), 2.73 (m, 2H

[CH2]‐5’), 3.73 (q, J = 4.5 Hz, 4H [2CH2]‐2’3’), 3.81‐3.89 (q, J = 10.5 Hz, 4H, [N(2CH2)]‐1’,4’),

3.97 (s, 2H [CH2]‐8’), 4.05‐4.17 (m, 1H, [N(CH)]‐1), 7.72 (d, J = 6 Hz, 1H, [CH]‐8), 8.04 (d, J =

3 Hz, 1H [NH]‐5)., 8.26 (s, 1H, [CH]‐2), 8.78 (s, 1H, [COOH]‐2).13C‐NMR (300 Hz, DMSO‐d6): δ

7.97 (d, [N(CH2CH3)]‐1), 25.83 (C‐3”,4”), 29.19 (C‐6’,7’), 34.34 (C‐5’), 36.32 (C‐8’), 39.31

([N(CH2CH3)]‐1), 40.74 (C‐2’3’), 49.45 (C‐1’,4’), 107.11 (C‐8), 111.51 (C‐3), 119.29 (C‐5),

139.46 (C‐9), 145.30 (d, J = 10.5 Hz, C‐10), 148.43 (C‐2’), 161.43 (C‐7), 166.27 (C‐6), 172.59

(C‐3), 173.94 (C‐2”,5”), 176.70 (C‐4).ESI‐HRMS: calculated for C25H29FN4O6: 500.21, found

m/z 501.2142 [M+H]+.

59

Scheme 3. Synthesis of compound 3 (4‐bromobutyl NHS ester)

Scheme 4. Synthesis of compound 4 (denoted as ciprofloxacin butyl NHS ester hapten)

3.3.3 The spacer arm attachment via piperazinyl moiety of norfloxacin to form

norfloxacin crotyl NHS ester

Norfloxacin crotyl NHS ester hapten was derived from the indirect conjugation of a

bromocrotyl NHS ester to the piperazynil group on norfloxacin. Initially, 4‐bromocrotonic

acid was prepared by hydrolysing 4‐bromocrotonate with dilute sodium hydroxide solution

and acidifying with diluted hydrochloride acid solution to give compound 5 (scheme 5). To

60

form a bromocrotyl NHS ester linker, 4‐bromocrotonic acid was reacted with DCC and NHS

in dimethoxy ethanol and DME to give compound 6 (scheme 6). Bromocrotyl NHS ester

was then attached through the piperazinyl group of norfloxacin in the presence of NHS to

make norfloxacin crotyl NHS ester hapten, compound 7 (scheme 7). Product was washed

with water and extracted with ethyl acetate. No further purification was performed.

3.3.3.1 Synthesis of 4‐bromocrotonic acid compound (5), scheme 5

Sodium hydroxide solution (1M, 10 mL) was added to methyl 4‐bromocrotonate (3000 mg,

17.0 mmol). The mixture was stirred at 0‐5oC for 5 h and left to stand at 4oC for 12‐24 h.

The mixture was acidified with hydrochloride acid (1M, 20 mL) and extracted with ether (3

x 25 mL). The organic layer was dried with anhydrous sodium sulphate and evaporated to

dryness under high vacuum. The product was purified by a flash column chromatography

(i.e. packed with silica gel; column size; diameter: 30 mm, length: 20 cm; flow rate: 1

mL/min; eluent: ethyl acetate/MeOH = 19:1) and the fraction containing the product was

evaporated to dryness under vacuum, giving a light yellow solid product (850 mg, 30%). 1H‐

NMR (300 Hz, CDCl3): δ 4.03 (dd, J= 3 Hz, 2H, [(CH2)Br]‐4), 6.05 (dt, J = 3 Hz, 15 Hz, 1H,

[(CH)]‐2), 7.07‐7.17 (m, 1H,[(CH)]‐3).

3.3.3.2 Synthesis of bromocrotyl NHS ester linker, compound (6), scheme 6

NHS (430 mg, 5.7 mmol) and DCC (1189 mg, 5.7 mmol) were added to a solution of 4‐

bromocrotonic acid (850 mg, 5.15 mmol) in DME (4 mL). The mixture was stirred at 0‐5oC

for 5 h and allowed to stand at 4oC for 12‐24 h. The product was purified by a flash column

chromatography (silica; column size; diameter: 30 mm, length: 20 cm; flow rate: 1 mL/min;

DCM/n‐hexane = 1:2) and evaporated to dryness under vacuum, giving a white solid (1000

mg, 41%). 1H‐NMR (300 Hz, CDCl3):δ 2.86 (s, 4H [(2CH2)]‐2’,3’), 4.07 (dd, J= 3 Hz, 2H,

[(CH2)Br]‐4), 6.25 (dt, J = 3 Hz, 15 Hz, 1H, [(CH)]‐2), 7.23‐7.33 (m, 1H, [(CH)]‐3).

3.3.3.3 Synthesis of (E)‐7‐(4‐(4‐((2,5‐dioxopyrrolidin‐1‐yl)oxy)‐4‐oxobut‐2‐en‐1‐

yl)piperazin‐1‐yl)‐1‐ethyl‐6‐fluoro‐4‐oxo‐1,4‐dihydroquinoline‐3‐carboxylic acid

[Norfloxacincrotyl NHS ester hapten], compound (7), scheme 7

To a dry DMSO (5mL), sodium hydride (NaH; 25.4 mg, 1.06 mmol) was added and stirred at

room temperature (approximately 25oC) for 10min. Norfloxacin (200 mg, 0.6 mmol) was

added and the mixture was stirred at room temperature for another 10 min. Bromocrotyl

NHS ester was then added and the mixture was stirred at room temperature for 24 h. The

61

mixture was washed with water (25mL) and extracted with ethyl acetate (4 x 20mL). The

organic layer was evaporated to dryness under vacuum, giving a brown solid product (100

mg, 50%), mp = 170‐174oC (decomposition). 1H‐NMR (300 Hz, CDCl3): δ 1.60 (q, J = 7.5, 3H,

[N(CH2CH3)]‐1), 2.62 (s, 4H, [(2CH2)],‐3”,4”), 2.72 (t, J = 4.5 Hz, 4H, [(2CH2)]‐2,3‐piperazinyl),

3.36 (t, J = 4.5 Hz, 2H, [(CH2)]‐1,4‐piperazinyl), 4.22 (q, J = 9 Hz, 2H, [(CH2)]‐4’), 4.34 (q, J =

7.5 Hz, 2H [N(CH2)]‐1), 6.05, (dd, J = 15 Hz, 1H [(CH)]‐2’), 6.85 (d, J = 9 Hz, 1H [(CH)]‐8),

6.93‐7.02 (m, 1H, [(CH)]‐3’), 8.04 (d, J= 12 Hz, 1H, [(CH)]‐5,), 8.68 (s, 1H, [(CH)]‐2). 13C‐NMR

(75 Hz, CDCl3): δ 14.35 (d, J = 15 Hz, [N(CH2CH3)]‐1), 40.97 (C‐3”,4”), 49.83 (d, J = 5.25 Hz,

[N(CH2CH3]‐1), 52.81 (C‐1,4‐piperazinyl), 59.00 (C‐2,3‐piperazinyl), 60.52 (C‐4’), 103.83 (C‐8),

108.34 (C‐3), 112.62 (C‐5), 120.579(d, J = 7.5 Hz, C‐9), 123.83 (C‐2’), 137.11 (C‐10), 144.24

(C‐3’), 146.05 (C‐7), 147.13 (C‐2), 151.86 (C‐6), 155.19 (COOH), 166.11 (C‐2”,5”), 166.24 (C‐

1’), 176.96 (d, J = 3 Hz, C‐4).

Scheme 5.Synthesis of compound 5 (4‐bromocrotonic acid).

Scheme 6.Synthesis of compound 6 (bromocrotyl NHS ester).

62

Scheme 7. Synthesis of compound 7 (denoted as norfloxacin crotyl NHS ester hapten).

3.4 Result and discussion

3.4.1 Hapten selection and synthesis

Two points of attachment of a linker can be considered for designing FQ haptens; 1) the

carboxylic acid group and 2) the piperazinyl group (Figure 3.2). From the antibody

production view, attaching a linker to a carboxylic acid group will lead to enhanced

specificity of the antibody towards its specific target. This approach helps to extend the

variable moiety away from the point of attachment to a carrier protein, which may lead to

antibodies that have better recognition for the variable portion of the molecule. While

conjugating a linker to the piperazinyl group and exposing the carboxylic acid group will

lead to antibodies with cross reactive because the moiety that is extended away from the

point of attachment would be part of the FQ backbone structure.

Almost all of the previous studies focused on ENR hapten that was directly conjugated to a

carrier protein without a linker (Wang et al., 2007b, Bucknall et al., 2003, Kato et al., 2007,

Cao et al., 2009). The assay resulted antibodies having broader cross‐reactivity with other

FQ drugs. In this study, a four carbon linker of tert‐butyl β‐alanine was attached to the

carboxylic group of ENR, producing a compound denoted as tert‐butyl ENR. The tert‐butyl

63

group was then cleaved by TFA to give ENR acid hapten containing a carboxylic acid on its

structure. The purpose of a linker was to provide some distance between the hapten and

the carrier protein and give better exposure of the whole structure to immune system. It

was postulated that the polyclonal antibodies produced against this hapten may

differentiate ENR from other FQ drugs, i.e., the cross‐reactivity with the related FQ

compounds would be significantly reduced.

Meanwhile, the ciprofloxacin butyl NHS ester hapten was successfully synthesised with 4‐

bromobutane NHS ester containing four carbons to the piperazinyl group of ciprofloxacin,

and was expected to generate class specific antibodies. Another FQ hapten successfully

synthesised was the norfloxacin crotyl NHS ester hapten. Bromocrotyl NHS ester was

attached to the piperazinyl moiety of norfloxacin and was also expected to produce a

generic ELISA for detecting multiple targets of FQs.

3.4.2 ENR acid hapten synthesis

FQ antibiotics are soluble particularly in polar organic solvents (i.e. methanol and

dichloromethane) and are soluble in hydro‐organic or aqueous acidic and basic buffer, but

are not soluble in non polar solvents (Brown, 1996). The solubility of FQs base form was an

issue in hapten synthesis. To increase solubility of the target FQs, FQ salts (ENR HCl,

ciprofloxacin HCl and norfloxacin HCl) were prepared as well as obtained from commercial

sources i.e., Ruland Chemistry Company Ltd., for comparison with synthesised standards.

Many FQs such as ENR are very sensitive to moisture, light and temperature. Hence, ENR

hapten synthesis was carried out at low temperatures, in dark and in anhydrous conditions

wherever possible.

3.4.2.1 Synthesis of tert‐butyl 3‐(1‐cyclopropyl‐7‐(4‐ethylpiperazin‐1‐yl)‐6‐fluoro‐4‐oxo‐

1,4‐dihydroquinoline‐3‐carboxamido)propanoate, ENR tert‐butyl

Figure 3.3 The chemical structure of tert‐butyl ENR hapten.

64

Tert‐butyl ENR strongly interacted with silica (<20% recovery) and upon interaction it was

very susceptible to degradation on silica chromatography. Initial attempts to purify tert‐

butyl ENR were performed by flash column chromatography using silica gel. The 1H‐NMR

spectrum of the purified tert‐butyl ENR indicated that it decomposed since there was no

cyclopropyl group on the primary amine at 4.18 ppm, no aromatic group at 7.03 and 8.29

ppm, and no tert‐butyl group at 1.38 ppm (Figure 3.6). The purification was then optimized

using a basic eluent system consisting of a mixture of ethyl acetate and triethylamine (TEA)

to neutralize the acidity of the silica gel on a flash chromatography system. However, this

condition did not prevent degradation of the product. To overcome this problem,

preparative thin layer chromatography (PTLC) was carried out, but was unsuccessful.

Consequently, conjugation of the ENR hapten to a carrier protein via two step reactions

was conducted without purification of the NHS active ester.

After putting considerable efforts on silica column without much success, gravity column

chromatography using neutral alumina was investigated, using ethyl acetate as an elution

solvent. Characterization by 1H‐NMR, as shown in Appendix A, confirmed an intact tert‐

butyl ENR hapten with no significant decomposition. A signal at 1.86 ppm corresponded to

the tert‐butyl group containing nine hydrogen atoms. More characteristic signals were a

doublet at 7.93 ppm that corresponds to the proton at position 2” and the quartet signals

at 3.95 and 4.48 ppm that belong to the tert‐butyl side chain at position 4” and 3”,

respectively. However, a dicyclohexylcarbodiimide urea by product was still visible in the

NMR spectra, showing several broad peaks with multiplets at 1.62 to 2.46 ppm. The signals

of dicyclohexylcarbodiimide urea by‐product usually can be detected at around 1.70 ppm

(multiplet), 2.46 and 3.38 ppm (triplet) and 2.26 ppm (single), which corresponds to six

hydrogen atoms on its structure (Mock and Ochwat, 2002). The singlet at 2.44 ppm was

also present on the tert‐butyl ENR1H‐NMR spectrum. This impurity, however, was not

removed to avoid potential decomposition of the product. 13C‐NMR and 135 DEPT‐NMR

characterizations were also performed to confirm the backbone of tert‐butyl ENR

(Appendix B and C).

65


dihydroquinoline‐3‐carboxamido)propanoic acid, ENR acid hapten

Tert‐butyl ENR was treated with TFA to form the ENR butyric acid hapten (Figure 3.8). The

cleavage of tert‐butyl was confirmed to occur during TLC as determined using 1H‐NMR, 13C‐

NMR and LC/MS. Therefore, no purification was performed

Figure 3.4 The chemical structure of ENR acid hapten.

Based on the TLC, there were three spots which were subsequently eluted with ethyl

acetate namely, ENR, tert‐butyl ENR, and ENR acid hapten. All spots were intensely visible

under UV light at different Rf. The 1H‐NMR spectrum of ENR acid hapten (Appendix D)

confirmed the absence of hydrogen atoms belonging to a tert‐butyl group (9H, 3CH3),

which indicated the successful cleavage of the tert‐butyl group. Other characteristic signals

determined using1H‐NMR were the number of protons that correspond to the side chain of

ENR acid hapten structure at positions 2”, 3” and 4”. Further confirmation was conducted

using 13C‐NMR and 135 DEPT‐NMR to evaluate ENR acid hapten (Appendix E and F).

As shown in Appendix G, low resolution mass spectrometry (LRMS) confirmed the

formation of ENR acid hapten, with the m/z 431.30 [M + Na]+ being consistent with

C22H27FN4O4. An exact mass measurement verified the presence of m/z peaks

corresponding to the molecular weight of ENR acid hapten of 430.47.

3.4.3 Ciprofloxacin bromobutane NHS ester hapten synthesis

Another attachment point for a spacer arm was used for the ciprofloxacin hapten synthesis

which was through a piperazinyl group of the FQ structure. This hapten was expected to

generate a class specific assay by exposing a carboxylic moiety as an immunodominant

region of FQs structures. To prepare ciprofloxacin bromobutane NHS ester hapten, a 4‐

bromobutane NHS ester linker was first formed by an attachment to the piperazinyl group

of ciprofloxacin as described below.

66

3.4.3.1 Synthesis of (1‐(4‐bromobutoxy)pyrrolidine‐2,5‐dione), 4‐bromobutane NHS ester

linker

Figure 3.5 The chemical structure of 4‐bromobutane NHS ester linker.

1,4‐Dibromobutane was selected as a linker with four carbons. It proved to be sufficiently

reactive to a hydroxyl group of NHS to form an active ester. The final brown viscous

product was confirmed by TLC (ethyl acetate, Rf = 0.67) under UV light; no further

purification was conducted. There were three spot regions where compounds eluted,

namely 1,4‐dibromobutane, NHS and the ester linker. The TLC showed an intense spot at a

different Rf value indicated that 1,4‐dibromobutane was successfully linked to NHS. Further

characterization using1H NMR confirmed the identity of the product which was 4‐

bromobutane NHS ester (Appendix H).

3.4.3.2. Synthesis of (1‐cyclopropyl‐7‐(4‐(4‐((2,5‐dioxopyrrolidin‐1‐yl)oxy)butyl)

piperazin‐1‐yl)‐6‐fluoro‐4‐oxo‐1,4‐dihydroquinoline‐3‐carboxylic acid), ciprofloxacin

butane NHS ester hapten

Figure 3.6 The chemical structure of ciprofloxacin butane NHS ether hapten.

Once 4‐bromobutane NHS ester was formed, it was reacted to the piperazinyl moiety of

ciprofloxacin to give a ciprofloxacin hapten. A few variants of the experiment were carried

out to form ciprofloxacin hapten using triethylamine (TEA), anhydrous potassium

carbonate (K2CO3) or sodium hydrate (NaH) as catalysts. The first reaction using DMF as a

solvent and TEA as a catalyst to form ciprofloxacin hapten (Figure 3.9) failed to allow

bromobutane NHS ester to react with ciprofloxacin. Also, there was a breakdown of

piperazinyl moiety at position C‐1’ to C‐5’ of ciprofloxacin.

67

Figure 3.7 Synthesis of ciprofloxacin butane NHS ether hapten catalysed by TEA.

A mild catalyst, K2CO3, was attempted to form a ciprofloxacin haptensinceK2CO3 was

successfully used in the reaction of 4‐bromobutane NHS ester. The reaction (Figure 3.10)

was again periodically monitored by TLC and 1H‐NMR, but the 1H‐NMR spectrum indicated

no success. Therefore, a more harsh condition using NaH as a catalyst and dry DMSO was

conducted. NaH is a strong base and reacts easily with an amine of the piperazinyl moiety.

The TLC showing an intense spot at a different Rf value indicated that 4‐bromobutane NHS

ester was successfully linking to ciprofloxacin to form ciprofloxacin bromo NHS ester

hapten

68

Figure 3.8 Synthesis of ciprofloxacin bromo NHS ether hapten catalysed by K2CO3.

Further characterization by 1H‐NMR showed that proton corresponded to proton at

position C‐5’ which was the triplet at 2.73 ppm (methylene group attached to piperazinyl

moiety), and other protons that corresponded to the five member ring of ester linker at

position C‐6’, C‐7’, C‐8’ were present (Appendix I). However, the proton at position C‐8’

(methylene group attached to oxygen of the ester linker) showed a broad singlet at 3.97

ppm, which was expected to be a triplet. This was likely due to the proximity to an oxygen

group resulting in merging of anion and thus broadening of the signal. The13C‐NMR

spectrum also confirmed the backbone of ciprofloxacin hapten. The ESI‐HRMS (Appendix L)

confirmed the presence of m/z peaks corresponding to the molecular weight of

ciprofloxacin butane NHS ester hapten of 500.52.

3.4.4 Norfloxacin crotyl NHS ester hapten synthesis

Norfloxacin crotyl NHS ester hapten was formed by attaching bromocrotyl NHS ester to the

piperazinyl group of norfloxacin so that the spacer arm could then be attached. Norfloxacin

hapten was synthesised using bromocrotyl NHS ester as a linker to develop a broad cross

reactive assay. Bromocrotyl NHS ester was chosen because it was an active electrophile,

and was sufficiently reactive to an amine on the piperazinyl group of norfloxacin. Also,

69

bromocrotyl NHS ester yields four carbon in length, which is considered ideal as a spacer

arm. Presence of a double bond in a bromocrotyl NHS ester usually is not preferred.

However, a double bound on its structure would increase binding differentiation when

used as a competitor (hapten‐protein conjugate) in a competitive assay, therefore may

lead to better sensitivity towards the target analyte.

3.4.4.1 Synthesis of 4‐bromocrotonic acid

Figure 3.9 The chemical structure of 4‐bromocrotonic acid.

4‐Bromocrotonic acid was prepared by alkaline hydrolysis of methyl 4‐bromocrotonate at

0oC. The final product was characterized by 1H‐NMR and the spectrum is shown in

Appendix M. In the 1H‐NMR spectrum, signals at 6.05 and 7.07‐7.17 ppm which

corresponded to the double bond at position 2 and 3, respectively, were present. Also, a

doublet of doublet at 4.03 ppm corresponded to the proton at position C‐4 (methylene

group attached to bromine).

3.4.4.2 Synthesis of bromocrotyl NHS ester linker

Figure 3.10 The chemical structure bromocrotyl NHS ester.

Once 4‐bromocrotonic was formed, it was reacted with NHS and DCC at 0oC to yield a

bromocrotyl NHS ester. The white solid product was characterized by 1H‐NMR (Appendix

N).The signals at 6.25 ppm and 7.23‐7.33 ppm belonging to the double bond, and the

proton at 4.07 ppm were present. In addition to that, a singlet at 2.86 ppm corresponded

to the protons of the five member ring of the linker.

70

3.4.4.3 Synthesis of norfloxacin crotyl NHS ester hapten

The1H‐NMR spectrum of the final product of norfloxacin hapten indicated that the ester

linker was attached (Appendix O). This is evidenced by the presence of a quartet at 4.11

ppm, the protons corresponded to the methylene group attached to an amine on

piperazinyl group of norfloxacin. The protons belonging to the double bond at 6.05 and

6.80‐6.89 ppm and the five member ring of the NHS moiety at 2.59 ppm were present.

Furthermore, 13C‐NMR and 135 DEPT‐NMR confirmed the backbone of norfloxacin hapten,

as shown in Appendix P and Q.

3.5 Conclusion

In this study, three new FQ haptens were synthesised, namely ENR acid, ciprofloxacin

butane NHS ester and norfloxacin crotyl NHS ester haptens, using different attachment

point of the linkers onto the FQ structures (i.e., carboxylic acid or piperazinyl group). A

carboxylic acid group was attached with a β‐alanine linker in synthesising ENR acid hapten.

Whereas, piperazynil moiety was attached with a 4‐bromobutane NHS ester linker and

bromocrotyl NHS ester linker in forming the ciprofloxacin butyl NHS ester and norfloxacin

crotyl NHS ester haptens, respectively. In regard to the antibody production, ENR acid

hapten‐protein conjugate was expected to generate antibodies for a specific assay. While,

ciprofloxacin butane NHS ester‐protein and norfloxacin crotyl NHS ester‐protein conjugates

were expected to produce antibodies for a generic assay (broad specificity).

The reaction condition and purification method were optimised for FQs hapten synthesis.

Selection of suitable solvents, performing at ambient temperature, in the dark and

anhydrous conditions and using gravity column purification method using alumina were

crucial parts for the successful synthesis of ENR hapten.

Ciprofloxacin and norfloxacin haptens were carried out employing more harsh condition

(i.e. NaH catalyst and reflux). The linkers containing a four‐carbon atom were firstly

synthesised to prepare ciprofloxacin and norfloxacin haptens. The 4‐Bromobutane NHS

ester linker was conjugated to the piperazynil group on ciprofloxacin to form the

ciprofloxacin butyl NHS ester hapten. Also, the bromocrotyl NHS ester linker was linked to

the piperazinyl group of norfloxacin to produce norfloxacin crotyl NHS ester hapten.

71

CHAPTER 4. DEVELOPMENT OF THE SPECIFIC ENR ELISA (ENR‐ELISA)

4.1 Introduction

ENR (ENR) is a member of the FQ family of antibiotics, with high bactericidal action. It was

the first FQs drug used in veterinary medicine and for pets and domestic animals treatment

since 1995 (Nelson et al., 2007). It is also used frequently in cattle and poultry to eliminate

Salmonella infection (Mitchell, 2006). However, due to increases in the resistant strain of

Campylobacter sp., it has been withdrawn from the U.S. market in 2005, (Nelson et al.,

2007), Zhao et al., 2009). The use of ENR as a veterinary drug in many countries (other than

the U.S.) leads to the concern about the presence and accumulation of ENR residues in

food. Residues of ENR in animal edible tissues entering into the human food chain could

pose a serious hazard to human health and a potential risk for an emergence and spread of

FQ resistant bacteria (Sheng et al., 2009a).

To prevent the negative impact of FQ residues on human health, the maximum residue

limits (MRLs) of FQs in seafood and animal‐derived products have been established and

regulated by various agencies such as the European Union (EU) and, and the Regulation for

Usage Veterinary Medicines in Japan. The EU and the joint FAO/WHO Expert Committee on

Food Additives (JECFA) have established MRLs for ENR, ciprofloxacin and their active

metabolites between 100 and 300 µg kg‐1 in muscle tissues, fat and milk for all species

(Hernandez‐Arteseros et al., 2002). The National Standardization Agency of Indonesia

refers to FAO/WHO Expert Committee on Food Authority (JECFA) for guidance in

establishing MRLs for FQ residues. Meanwhile, in Australia, FQ antibiotics are not

permitted for use in agriculture or aquaculture without a specific permit or prescription.

In order to regulate FQs residues in foodstuffs of animal origin, a wide range of analytical

methods have been applied. Analytical methods used for the determination of FQs

residues include high performance liquid chromatography (HPLC) with UV/fluorescence

detectors (Hung, 2007, Carlucci, 1998, Espinosa‐Mansilla et al., 2006, Gigosos et al., 2000,

Hassonuan et al., 2007a, Hassonuan et al., 2007b, Holtzapple et al., 2000, Yorke and Froc,

2000, Zheng et al., 2005, Holtzapple et al., 2001, Holtzapple and Stanker, 1998, Pena et al.,

72

2010), liquid chromatography mass spectrometry (LC‐MS) (Marchesini et al., 2007), liquid

chromatography tandem mass spectrometry (LC‐MS/MS) (Bogialli et al., 2008, Ikegawa,

1998, Johnston et al., 2002, Toussaint et al., 2005a, Toussaint et al., 2005b, Van Vyncht et

al., 2002), capillary electrophoresis (Fierens et al., 2000) gas chromatography (GC)

(Takatsuki, 1991), thin layer chromatography (TLC) / immuno‐chromatography (Sun et al.,

2007, Zhu et al., 2008). More recently, bio sensors for FQ detection, which require no

separation steps, have been reported (Cao et al., 2007, Giroud et al., 2009, Marchesini et

al., 2007).

These instrumental methods often required extensive sample preparation that involves

interference removal and concentration, which is a time‐consuming and laborious process.

These methods also require technically skilled personnel to maintain instruments and

interpret instrument outputs. Moreover, many of these instruments are very expensive in

both setting up and running costs. Therefore, developing simple, rapid and cost‐effective

methods, such as immunoassays, that provide advantages over the instrumentation

techniques would be beneficial for laboratories that cannot afford such expensive

instruments, such as those regulatory laboratories in Indonesia.

Immunoassays are a method principally based on antibody‐antigen binding properties.

They have been applied to determine a wide range of contaminants, including pesticides

and veterinary drugs in food. The technique increasingly play an important role in food

analysis by improving analytical capacity, thus ensuring food safety (Chen et al., 2009b).

Many immunoassays as screening methods also have been successfully used as

alternatives or complementary to instrument‐based methods for detecting FQs

contaminants in food commodities ( Bucknall et al., 2003, Huet et al., 2006, Wang et al.,

2007a, Li et al., 2008).

Most of the ENR immunoassays also detect other FQs to certain degrees (Wang et al.,

2007b, Bucknall et al., 2003, Kato et al., 2007, Cao et al., 2009). To our best knowledge,

there have been no reports on an assay that is highly specific to ENR. Hence, this study

reports, for this first time, the synthesis of novel haptens of ENR with a suitable spacer arm

and the development of an indirect competitive ELISA highly specific to ENR. The validation

73

of the developed assay against potential interference from animal or marine derived

products, and analytical performance using spike and recovery study are also reported.

4.2 Materials and methods

4.2.1 Materials and Instrumentation

4.2.1.1 Materials

Bovine serum albumin (BSA), ovalbumin (OA), keyhole limpet hemocyanin (KLH), and

horseradish peroxidase (HRP) were purchased from Sigma Aldrich, US. Dialysis tubing,

Tween 20, secondary goat anti‐rabbit IgG‐HRP antibody, and Freund incomplete adjuvants

were also bought from Sigma Aldrich, US. Salts of FQ antibiotics were obtained from

Ruland Chemistry Company Ltd., China. FQ antibiotics were bought from Sigma Aldrich,

USA. Maxisorp polystyrene 96‐well microtitre plates were from Nunc, Denmark. Silica gel H

was obtained from Merck (Darmstadt, Germany) and deuterated solvents for NMR such as

dimethylsulfoxide, chloroform and water were obtained from Cambridge Isotope

Laboratories (CIL), Inc. (US).

4.2.1.2 Instruments

Antibody concentrations and immunoassay absorbance were measured by SpectraMax®

M2, a multi‐detection microplate reader from Molecular Devices (Sunnyvale, California,

USA). A digital pH meter was obtained from TPS Pty. Ltd. (Brisbane, Australia).

4.2.2 Antibody production and characterization

4.2.2.1 Preparation of conjugates of hapten and carrier proteins or enzyme

For the antibody production, immunogens were prepared by conjugating the synthesised

haptens (in active ester forms) to the carrier protein, KLH. For ELISA, antigens are prepared

by conjugating haptens to BSA or OA. All conjugations were performed via the

carbodiimide/NHS active ester method.

Coupling protocol: ENR hapten (ENR1, 0.03 mmol) was dissolved in 1 mL of dried DMF,

containing DCC (0.04mmol) and NHS (0.04mmol) before adding to the ENR‐1 solution. The

precipitate of the by‐product, dicyclohexyl urea, was filtered through a cotton filter, leaving

a clear supernatant for the subsequent conjugation. After the filtration, the ENR active

74

ester solution was added dropwise to each protein (BSA, OA or KLH) dissolved in pre‐

cooled coupling buffer (50mM phosphate buffer, pH 9.1). The schematic reaction is shown

in Figure 4.1. The reaction solution was gently stirred and was allowed to stand at 4C

overnight. The conjugated solution was then dialysed against 50mM phosphate buffered

saline (PBS, pH 7.4) with several buffer changes and the protein conjugate solution was

stored at 4C until use.

Figure 4.1 The schematic reaction of ENR hapten‐protein conjugation.

4.2.2.2 Immunisation and antibody production

An immunogen was prepared by emulsifying ENR‐1‐KLH with 0.9% NaCl (saline) and

TitreMax Gold adjuvantor Freund’s incomplete adjuvant. The New Zealand white rabbits

were immunised with the immunogen by subcutaneous injections. Subsequent booster

injections were given at monthly intervals for six months. The blood was collected from the

marginal ear vein on a monthly basis and the antiserum was isolated from the blood by

centrifugation.

75

4.2.2.3 Purification of Rabbit IgG

Sera were purified by an affinity chromatography on protein A/G Sepharose (Pharmacia‐GE

healthcare). Firstly, the serum was diluted with an equal volume of phosphate buffer and

the mixture was loaded onto the protein A/G column (protein A/G Sepharose,

size/volume/capacity = 5 mL, flow rate = 1.5 mL/min). The IgG was eluted with acetate

buffer at pH 4. The eluent which contained the IgG fraction was immediately neutralised

with 1 M Tris base (pH 11). The purified antibody was then dialysed against PBS, pH 7.4,

before storing at 4°C.

4.2.2.4 Determining antibody concentration

The concentration of purified polyclonal antibodies (pAbs) was determined by measuring

the absorbance at 280 nm. The absorbance was measured against PBS as a reference. The

concentration of antibody was calculated using Equation 1:

Antibody concentration (mg/mL) = 1........EqdA

Where: A = absorbance at 280 nm

d = dilution factor

ε = IgG extinction coefficient, which is 1.35 (Nikolayenko et al., 2005).

4.2.2.5 Determining optimum working concentration by checkerboard titration

The checkerboard titration (CBT) was performed to determine the optimum working

concentrations of each antibody and enzyme‐conjugate pair. The CBT was carried out as

follows:

1. A coating conjugate (e.g., either OA‐ or BSA‐hapten conjugate) was immobilised on a

96‐microwell plate by incubating a coating antigen at 10 µg mL‐1 (1 µg per well)

dissolved in the coating buffer (50mM carbonate buffer, pH 9.6). After the plate was

left to stand overnight at room temperature, it was washed for three times with

deionized water and dried by tapping on an absorbent paper towel.

76

2. For blocking of the unoccupied space in the microwells, the plate was incubated with 3%

skim milk solution at 200 µL per well for 1 h at room temperature, prior to being

washed three times with deionized water.

3. Antibodies were diluted in 1% fish gelatine in PBS (denoted as FG/PBS) and were

titrated in 3‐fold serial dilutions. The plate was incubated for 1h and washed as

previously described.

4. A solution of anti‐rabbit IgG‐HRP conjugate (1:2000 in 1% FG/PBS‐0.05% Tween 20, 100

µL per well) was incubated for an additional 1h at room temperature.

5. The colour reaction was developed by incubating with a substrate/chromogen solution

(1.25mM 3,3’5,5’‐tetramethylbenzidine(TMB)in acetate buffer (pH 5.0) containing urea

hydrogen peroxide, 100 µL per well) for 30 min.

6. The reaction was stopped by adding the stop solution (1.25M sulfuric acid, 50 µL per

well).

7. The absorbance was measured at 450 nm and 650 nm.

4.2.2.6 Determining Sensitivity

4.2.2.6.1 Preparation of standard solution

A 1000 µg L‐1 stock solution of ENR was prepared and diluted in 50 mM NaOH (0.5%) in PBS

(pH 7.4). A serial dilution of working standards at 1000, 300, 100, 30, 10, 3, 1, 0.3 and 0.1

ng L‐1 was prepared from the 1000µg L‐1stock solution.

4.2.2.6.2 Indirect Competitive ELISA protocol

The ENR‐ovalbumin conjugate (ENR‐OA) was immobilised onto a microwell plate at 1 ng

per well. The plate was washed three times with deionized water and dried. The unbound

sites on the microtitre plates were blocked with 3% skim milk (200 µL of per well) by

incubating for 1 h at room temperature, prior to washing and drying of the plate.

For the blank, the respective wells were loaded with 100 µL of deionized water and100 µL

of 50mMNaOH (0.5%) in PBS (pH 7.4). For the control, the respective wells were loaded

with 100 µL of 50mMNaOH 0.5% in PBS and 100 µL of 1% FG/PBS (as an antiserum diluent).

ENR standard solutions (100 µL per well) were added to the respective wells, followed by

77

an antiserum solution (100 µL per well). The mixture was incubated for 1 h at room

temperature. The microwells were washed again and a solution of anti‐rabbit IgG‐HRP

conjugate solution was incubated for 1h (100 µL per well). After washing the plate, colour

reaction was developed by incubating the substrate solution for 30 min and the reaction

was stopped with1.25M H2SO4. The absorbance was measured at 450nm and 650 nm.

Figure 4.2 illustrates the above procedure diagrammatically.

Figure 4.2 Schematic presentation of an indirect competitive ELISA for ENR.

4.2.2.6.3 Determination of standard curve parameter

A calibration curve (dose‐response) of an analyte was constructed by plotting

concentration on the x‐axis versus absorbance on the y‐axis. Percent inhibition (%I) for

each standard point and sample were calculated according to the Equation 2.The assay

sensitivity, calculated as an IC50, and a limit of detection (LOD), were obtained from the

calibration curves. An IC50 is a concentration of an analyte required to inhibit antigen‐

antibody interaction by 50%. The LOD for the assay was determined by a concentration

that reproducibly producing 20% inhibition of colour development.

78

4.2.2.7 Optimisation of ENR ELISA conditions

4.2.2.7.1 The effect of antiserum diluents

Tween 20 is commonly used in an assay diluent buffer to reduce non‐specific interactions,

especially for the synthetically prepared hapten‐protein conjugates. In order to optimise

ELISA conditions using Tween 20 as a buffer additive to reduce non‐specific binding, three

diluent solutions were examined; 1%FG/PBS, 1%FG/PBS+0.05%Tween 20, and 1%FG/PBS

+0.1%Tween 20.

4.2.2.7.2 The effect of organic solvents

Aqueous solubility of analytes could affect immunoassay performance. Fluoroquinolone

antibiotics are only soluble in certain solvents, such as methanol, and highly acidic and

basic solutions. Therefore, several solvent systems, containing acetone, acetonitrile,

methanol and ethanol between 5% and 20% in aqueous solutions, and mixtures of

methanol with glacial acetic acid or NaOH were tested to improve their aqueous solubility

and still maintain compatibility with an ELISA.

4.2.2.7.3. The effect of buffer solutions (pH conditions)

The stability of antibodies in different pH conditions, how pH may influence ionic states of

ENR and antibody binding affinity towards different ionic states of ENR were studied.

Buffer solutions with different pH ranging from 5.5 to 9.6 were used in this study. The pH

5.5 was made from citrate buffer, buffers at pH 6.5, 7.4, and 8.5 were obtained from PBS

and a buffer at pH 9.6 was prepared from carbonate buffer. All buffer solutions were

adjusted to the desired pH with either 1M HCl or NaOH solution.

4.2.2.8 Determining specificity

Assay specificity was evaluated using a set of fluoroquinolone antibiotics, i.e., norfloxacin,

ciprofloxacin, sarafloxacin, pefloxacin, nalidixic acid, enoxacin, and danoxacin (ranging from

0.1 to 100,000 µg L‐1). Assay specificities are expressed by the degree of cross‐reactivity of

an antibody to similarly structured compounds. Cross‐reactivity is calculated as the ratio of

an IC50 of the test compound and an IC50 of ENR (Equation 4).

79

4.2.2.9 Study of Matrix Effects

An ELISA is usually affected by the physical, biological, and chemical components in the

matrices such as protein, fat, sugar, pigments and tannins (Lee and Kenney, 2007). To

minimise matrix interferences, a number of techniques, such as extraction of an analyte

into a water miscible solvent, dilution, preheating to denature protein or a combination of

these techniques, was employed. As ENR is frequently used in farm animal and aquaculture,

edible tissue and animal‐derived products, such as liver, milk and prawn were chosen for

matrix effect studies.

4.2.2.9.1 Animal and marine product samples

Animal‐derived products (such as milk and chicken liver) and seafood products (such as

prawn) were obtained from various local sources, from Coles supermarket, local markets

and butchers in Kensington, Kingsford and Randwick, New South Wales. Milk samples

consisted of full cream milk, full cream milk powder, skim milk and skim milk powder.

Samples were also obtained from organic food markets (e.g., chicken liver) and imported

sources (e.g., prawn).

4.2.2.9.2 Protocol for sample extraction of chicken liver and prawn

Chicken liver and prawn samples were homogenised using a homogeniser. Homogenised

chicken liver or prawn (2.0 g) was mixed with 2 mL of 10% 50 mM NaOH in MeOH in a 50

mL polypropylene centrifuge tube, prior to being vortexed for 2 min. The PBS (18mL) was

added to the sample mixture and the solution was vortexed for 30 sec. The sample was

allowed to stand for 30 min and they were placed in a water‐bath shaker at 40oC for 15 min.

The solution was centrifuged at 4500 rpm (2268 g) for 10 min. The supernatant was

transferred into another centrifuge tube, and the sample was re‐extracted with 15 mL of

the extraction buffer twice and centrifuged using the same condition. The supernatants

were combined. Together with the blank solution, the extracted samples were spiked with

ENR for the matrix effect study.

80

4.2.2.9.3 Protocol for matrix effect determination of milk

Milk samples were preheated to 80oC for 5 min; 30 min heating was required for skim and

full cream milk samples. Samples were diluted by 5, 10 and 20‐fold (v/v) with PBS and were

centrifuged at 4500 rpm (2268 g) for 10 min.

4.2.3 Spiking and recovery studies

The recovery was determined by spiking ENR into milk, chicken liver and prawn samples to

the final concentrations of 5, 10, 20 and 50 µg L‐1, and the spiked samples were analysed by

ENR1 ELISA using AbαENR1‐KLH against ENR1‐OA.

4.2.3.1 Protocol for spiking of chicken liver and prawn with ENR

Chicken liver and prawn samples were spiked with various concentrations of ENR above

and below the MRL values prior to the extraction. Homogenised samples were weighed

(approximately 2.0 g) and placed into a 50 mL polypropylene centrifuge tube. ENR (not

exceeding 100 µL for a 2 g sample) at 250, 500, 1000 and 2500 µg L‐1 were spiked into the

homogenised samples to give the final concentrations of 5, 10, 20 and 50 µg L‐1 in the test

samples. The spiked samples were allowed to stand for 30 min at room temperature

before proceeding with the extraction.

4.2.3.2 Protocol for spiking of milk with ENR

Milk samples were spiked with ENR to achieve concentrations above and below the MRL

values for milk (between 10 and 3000 µg L‐1). ENR (not exceeding 100 µL) was spiked to the

final concentrations of 5, 10, 20 and 50 µg L‐1. Samples were allowed to stand for 30 min at

room temperature prior to the thermal treatment.

Table 4.1 shows the concentrations of ENR that were spiked into food samples and dilution

factors that were applied to the sample extraction. The recovery was calculated by

interpolating the ENR concentration from a % inhibition calibration curve.

81

Table 4.1 The concentrations of ENR used to spike samples

Spike

No.

The concentration of

spiked ENR (µg L‐1)

Dilution

Factor

The final concentration of

ENR in samples

(µg L‐1)

Samples

1

2

3

4

50

100

200

500

1 : 10

5

10

20

50

Milk

1

2

3

4

250

500

1000

5000

1 : 50

5

10

20

50

Chicken

liver and

Prawn

4.3 Results and Discussion

4.3.1 Antibody production and optimal concentration of ENR1 antiserum

Titre is defined, in our laboratory, as a titration factor of an antiserum that produces

“detectable” colour above the background (i.e., 0.1‐0.2 absorbance unit). An optimum

concentration ofeach bleed was assessed by conducting a checker board titration (CBT)

against an immobilised antigen and selecting a concentration of an antiserum that yielded

approximately 1‐1.5 absorbance units.

82

Figure 4.3 Titration curve of AbENR1‐KLH against ENR1‐OA from six different bleeds (first

bleed to sixth bleed) by an indirect ELISA format.

The Figure 4.3 presents titration curves of antisera from the first to the sixth collection

(defined as bleed). Each antiserum was titrated against ENR1‐OA antigen in an indirect

ELISA to allow selection of the optimum serum dilutions for further characterization in a

competitive format. The optimum concentration of first bleed (B#1), second bleed (B#2),

third bleed (B#3), forth bleed (B#4), fifth bleed (B#5) and sixth bleed (B#6) were 1/15,000,

1/10,000, 1/10,000, 1/5,000,1/5,000 and 1/2500, respectively. As shown in Figure 4.3, the

first bleed of AbENR1‐KLH yielded the highest titre of ENR specific antibody with a gradual

decrease in the titre values of the subsequent bleeds. This was unexpected and may be due

to the hydrolysis of the immunogen ENR1‐KLH conjugate, resulting in cleavage of the

hapten from the carrier protein during the storage at 4oC. Further immunization with newly

prepared immunogens, however, did not improve the immune response of the animals.

4.3.2 Antibody characterisation

The coating antigens were prepared by conjugating various FQ haptens to OA via the NHS

active ester method, yielding eight different FQ haptens‐OA conjugates. To evaluate assay

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

500 5000 50000 500000 5000000 50000000

Absorban

ce (450 nm)

Antisera dilution factor

B#1

B#2

B#3

B#4

B#5

B#6

83

sensitivity of the heterologous system, FQ haptens with the linkers, such as ENR‐t‐butyl

(ENR2‐OA) and ciprofloxacin‐bromobutane (CIP2‐OA) were synthesised. Hapten heterology

was also evaluated using various FQs that are directly conjugated to the carrier proteins

(i.e., haptens with no linker). These were ENR‐OA (ENR1‐OA), ciprofloxacin‐OA (CIP1‐OA),

norfloxacin‐OA (NOR‐OA), sarafloxacin‐OA (SAR‐OA), pefloxacin‐OA (PEF‐OA), nalidixic acid‐

OA (NAL‐OA). Using the first bleed (B#1) of the ENR1‐KLH antibody, the sensitivity of

homologous (i.e., ENR1‐AO) versus heterologous systems were evaluated in an indirect

competitive ELISA format.

Figure 4.4 Titration curves of AbENR1‐KLH#1(from bleed#1) against the eight hapten‐

protein conjugates (PEF‐OA, SAR‐OA, ENR2‐OA, NOR‐OA, NAL‐OA, CIP2‐OA, CIP1‐OA and

ENR1‐OA).

As can be seen from Figure 4.4, the highest titre of AbENR1‐KLH#1and ENR1‐OApair,

suggested high specificity to ENR, evident by decrease in the titre values (30‐fold decrease).

To be more practical in an immunoassay, the dilution factor of the antiserum that resulted

in 1‐1.5AU should be >5,000. Below this figure, there is a higher likelihood of an elevated

background colour due to non‐specific binding of the serum constituents. Thus, the

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

500 5000 50000 500000

Absorban

ce (450 nm)

Antisera Dilution Factor

PEF‐OA

SAR‐OA

ENR2‐OA

NOR‐OA

NAL‐OA

CIP2‐OA

CIP1‐OA

ENR1‐OA

84

homologous assay utilising AbENR1‐KLH (antibody) and ENR1‐OA as a competing antigen

was selected for further characterisation.

4.3.2 Assay Sensitivity

Assay sensitivity was defined by running standard curves of ENR in the homologous assay

using AbENR1‐KLH and ENR1‐OApair. The seven antigens derived from FQs‐OA conjugates

againstthe first and second bleeds of AbENR1‐KLH (B#1 and B#2) were evaluated (Table

4.2). The homologous assay generated better sensitivity than heterologous system, with an

IC50 value of 15 µg L‐1. The antibodies from the first bleed (B#1, IC50= 15 µg L

‐1) exhibited

better sensitivity than those from the second bleed (B#2, IC50 = 100 µg L‐1). The assay with

the hapten homology and linker heterology (i.e., using ENR2‐OA as a competing antigen)

gave very poor sensitivity (>1000 µg L‐1). While the hapten heterologous assays which were

based on other FQs‐OA conjugates exhibited very low colour development and poor

sigmoidal dose‐response curves.

Table 4.2 The IC50 values (µg L‐1) of the assays based on the combination of 14 FQ hapten‐

OA conjugates competed with ENR for AbENR1‐KLH by the indirect competitive ELISA

OA‐Conjugates

(Coating antigens)

IC50(µg L‐1)

First Bleed (B#1) Second Bleed (B#2)

ENR‐1‐OA

ENR‐2‐OA

CIP‐OA

NOR‐OA

SAR‐OA

NAL‐OA

PEF‐OA

15

>1000

ND*

ND*

ND*

ND*

ND*

100

>1000

ND*

ND*

ND*

ND*

ND*

ND* the assay exhibited very low colour, and did not show the ideal standard curves.

In this study, the absolute homologous assays using a combination of AbENR1‐KLH‐#1 as

an antibody and ENR‐1‐OA as an immobilised antigen (i.e., both hapten and linkage are the

85

same for the immunogen and the competing antigen) gave the best sensitivity of 15 µg L‐1.

A calibration curve using ENR‐1‐BSA as a solid phase antigen also did not show an ideal

dose‐response relationship. This was probably due to an interaction between BSA with ENR,

resulting in less available competing hapten (or epitope) on ENR1‐BSA conjugates or free

ENR (analyte) for binding with the antibody. BSA has been reported to increase non‐

specific binding of ciprofloxacin, resulting in lower sensitivity and specific and sensitivity in

an immunoassay (Snitkoff et al., 1998. Duan and Yuan, 2001).

4.3.3 Evaluation of assay parameters (IC80, IC50, IC20 and maximum absorbance)

Nine point calibration curves of ENR are shown in Figure 4.5. The results were averaged

over 25 analyses carried out on different days. The % CV of % inhibition decreased as the

concentration of ENR increased as shown in Figure 4.6. This curve indicates a typical

precision of a sigmoidal dose‐response relationship of an immunoassay (Lee and Kennedy,

2007). As shown in Table 4.3, the IC50 value was 11.8± 1.7 µg L‐1 (%CV = 14%). An IC20 value

refers to, in this study, as the LOD, was 2.4 ± 0.4 µg L‐1 (%CV = 17%). An IC80 value, which

referred to a concentration of ENR to inhibit 80% of colour development and defined as

upper detection limit of the detection range, was 91.4 ± 6.1 µg L‐1 (%CV = 7%). The average

maximum absorbance was 1.6± 0.1, with a %CV of 6%. The %CV of the absorbance of nine

ENR concentrations ranged from 5 to 35% (Figure 4.6).

Table 4.3 Standard curve parameters and precision of ENR assay

Parameters Concentration

(µg L‐1) S.D.a %CVb

IC50

IC20

IC80

Maximum absorbance

11.7

2.4

91.41

1.6

1.7

0.4

6.1

0.1

14

17

7

6

a standard deviation

b%coefficient variation

86

Figure 4.5 Calibration curves for AbENR1‐KLH (average of 25 analyses) based on the

absorbance () vs ENR concentration and the % inhibition () vs ENR concentrationusing

the optimised concentrations of anti‐ENR antibodies and immobilised ENRantigenwith an

IC50 value of 11.8µg L‐1± 1.7 and LOD 2.4 µg L‐1± 0.4. ± represents standard deviation

Figure 4.6 %CV of the absorbance () and the % inhibition () based on an average of 25

analyses.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

Absorban

ce (450 nm)

% In

hibition

ENR (µg L‐1)

0

10

20

30

40

50

60

0.1 1 10 100 1000

% CV

ENR (µg L‐1)

87

Figure 4.7 Plot of IC20 (), IC50 () and IC80 () values of the 25 standard curves.. The

middle solid lines indicate the average values of IC20, IC50 and IC80. The dotted lines indicate

the upper and lower limits of standard deviation; SD.

4.3.4 Characteristics of ENR ELISA

4.3.4.1 Assay specificity

Wang et al., (2007a) and Bucknall et al., (2003) designed an immunogen for ENR using the

secondary amino group located on the piperazynil moiety of ENR as a point of attachment

with a carboxylic acid group on a carrier protein. This design of hapten‐protein conjugates

displayed the 3‐quinolinecarboxylic acid moiety which was the common structure of FQs.

As a result, the antibody raised showed higher cross‐reactivity with other FQ compounds.

Moreover, a substitution or an addition of an amine on FQs and secondary amines on some

of the FQs played an important role in generating a broad cross‐reactive assay. The major

changes at the secondary amine of sarafloxacin, for instance, only contributed to a five‐fold

decrease in the antibody binding to ENR (Holtzapple et al., 1997). Consequently, this assay

showed a high cross‐reactivity for some structurally related FQ drugs. Hence, in this project

which aimed to develop antibodies highly specific to ENR, the hapten was synthesised by

conjugating a carboxylic acid group of ENR with an amino group of a carrier protein. The

1.00

10.00

100.00

0 5 10 15 20 25

ENR (µg L‐1)

number of assay (25 analyses)

88

absence of a carboxylic acid group at position 3 would direct the immune response to

antibodies that are highly specific to ENR.

Inspection of the cross‐reactivity data provides clues to which immunodominant moieties

of the hapten/analyte. The specificity of the antibody was evaluated by the cross‐reactivity

study against seven structurally related FQs and quinolones. These are danofloxacin (DAN),

enoxacin (ENO), sarafloxacin (SAR), pefloxacin (PEF), nalidixic acid(NAL) ciprofloxacin (CIP),

and norfloxacin (NOR). As shown in Table 4.5, little cross‐reaction was observed for the test

compounds, indicating that AbENR1‐KLH was indeed highly specific for ENR, confirming

the titration results.

AbENR1‐KLH showed low cross‐reactivity to DAN probably due to its diazobicyclo moiety

located on the secondary amine that was different from ENR. AbENR1‐KLH also resulted

in low cross‐reaction to SAR and NAL because of their apparent structural differences. For

instance, the fluorophenyl ring on the primary amine group of SAR drastically reduced

conformational similarity with ENR and hence resulted in poor antibody binding.

Meanwhile for NAL, the absence of piperazynil moiety and consisting of a ring substitutes

with a methyl group also contributed to poor recognition by AbENR1‐KLH. Despite the

high degree of structural similarity between ENO, PEF, CIP and NOR to ENR, very low cross‐

reactivity against these compounds was observed with % cross–reactivity (%CR) values

being <0.1%. Only PEF showed a slight cross‐reaction of 1.3%, probably due to a similarity

in the substituted group on the piperazynil group (i.e., a methyl group on PEF versus an

ethyl group on ENR).

89

Table 4.4 The IC50 (µg L‐1) and cross reactivity (%CR) for FQs related compounds

Compound Chemical structure IC

parameters µg L‐1

%CR

(µg L‐1) mol L‐1

%CR

(mol L‐1)

Enrofloxacin

(ENR)

IC50 27.31 100 7.6 x 10‐8 100

IC20 4.53 100 1.3 x 10‐8 100

IC80 232.06 100 6.5 x 10‐7 100

Danofloxacin

(DAN)

IC50 >1000 0.1 7.8 x 10‐5 0.1

IC20 >1000 0.2 8.4 x 10‐6 0.1

IC80 >1000 0.1 4.8 x 10‐4 0.1

Enoxacin

(ENO)

IC50 >1000 <0.1 4.6 x 10‐4 <0.1

IC20 >1000 <0.1 7.0 x 10‐5 <0.1

IC80 >1000 <0.1 4.6 x 10‐3 <0.1

Pefloxacin

(PEF)

IC50 >1000 1.3 7.2 x 10‐6 1.2

IC20 >1000 0.3 1.0 x 10‐6 0.3

IC80 >1000 1.8 6.0 x 10‐5 1.2

90

Table 4.4The IC50 (µg L‐1) and cross reactivity (%CR) for FQs related compounds (continued)

Compound Chemical structure

IC

parameter

s

µg L‐1 %CR

(µg L‐1) mol L‐1

%CR

(mol L‐1)

Nalidixic acid

(NAL)

IC50 >1000 <0.1 0.64 <0.1

IC20 >1000 <0.1 3.9 x 10‐3 <0.1

IC80 >1000 <0.1 106.53 <0.1

Ciprofloxacin

(CIP)

IC50 >1000 <0.1 2.7 x 10‐4 <0.1

IC20 >1000 <0.1 1.9 x 10‐5 <0.1

IC80 >1000 <0.1 1.4 x 10‐3 <0.1

Norfloxacin

(NOR)

IC50 >1000 <0.1 6.9 x 10‐4 <0.1

IC20 >1000 <0.1 3.7 x 10‐5 <0.1

IC80 >1000 <0.1 4.2 x 10‐3 <0.1

Sarafloxacin

(SAR)

IC50 >1000 <0.1 6.2 x 10‐4 <0.1

IC20 >1000 <0.1 8.7 x 10‐5 <0.1

IC80 >1000 <0.1 3.6 x 10‐3 <0.1

4.3.4.2 Assay Optimisation

Immunoassay performance may be affected by surfactants, organic solvents, pH, sample

matrices or salt concentrations (Lee and Kennedy, 2007; Sheng et al., 2009b). The effects of

these variables on the assay were tested by analysing changes in the maximum absorbance

values at zero concentration of ENR (denoted as the maximum absorbance, Amax), and IC50

91

values of standard curves of diluents containing Tween 20, organic solvents and varying pH

values.

4.3.4.2.1 Effects of diluents

Tween 20 is a non‐ionic surfactant and is commonly added to assay buffers to minimise

non‐specific binding (Lee and Kennedy, 2007). In order to determine the effect of this

surfactant on assay performance, four serum diluents consisting of PBS, 1% FG‐PBS, 1% FG‐

PBS + 0.05% Tween 20, 1% FG‐PBS + 0.1% Tween 20 were evaluated. As can be seen in

Table 4.6, these diluents, except for PBS, did not affect significantly on the assay

performance, with regards to both assay sensitivity and colour development. Briefly,

addition of fish gelatine enhanced both colour development and assay sensitivity as

measured by %I. An addition of Tween 20 enhanced colour development slightly, but did

not alter the sensitivity.

Table 4.5 Effect of diluents on antibody’s sensitivity

Assay diluents PBS ± SD (n=3) 1% FG‐PBS (control) ± SD (n=3)

1% FG‐PBS + 0.05%Tween 20

± SD (n=3)

1% FG‐PBS + 0.1% Tween 20

± SD (n=3)

Absorbance

maximum (Amax) ^0.8 ± 0.01 1.3 ± 0.02 ^1.6 ± 0.02 ^1.6 ± 0.01

%CV (Amax) 1.6 1.2 1.3 0.4

IC50 (µg L‐1) ^18.9 ± 1.5 11.0 ± 3.4 *11.5 ± 0.8 *13.4 ± 0.7

%CV (IC50) 7.9 30.7 7.0 5.6

Each value represents the mean of triplicates (n=3) with standard deviation (SD). Statistical

analysis was calculated using t‐test (α = 0.05). *no significant difference with control (1%

FG‐PBS). ^significant difference with control (1% FG‐PBS).

When PBS was used alone as a serum diluent without 1%FG and Tween 20, the IC50 values

increased approximately by two‐folds and the Amax values decreased by almost 50%

compared with 1%FG‐PBS as a control. Diluents containing Tween 20 affected colour

development considerably, by showing 0.2‐0.3 AU increase in Amax values. However, there

was no difference among the IC50 values of the assay diluents with and without Tween 20.

92

Hence Tween 20 up to 0.05% was concluded to not significantly affecting the antibody and

antigen binding. From these results, 1%FG‐PBS was suggested to be the best diluent for

this assay.

Figure 4.8 Effects of diluents (PBS, 1% FG‐PBS, 1% FG‐PBS + 0.05% Tween 20 and 1% FG‐

PBS + 0.1% Tween 20) on colour development of the ELISA based on AbENR1‐KLH. Each

value represents a mean of triplicates (n=3) with a standard deviation (SD) value of PBS, 1%

FG‐PBS, 1% FG‐PBS + 0.05% Tween 20 and 1% FG‐PBS + 0.1% Tween 20 was 0.01, 0.02, 0.02

and 0.01, respectively. Statistical Analysis was calculated using t‐test ( = 0.05).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

PBS

1% FG‐PBS

1% FG‐PBS + 0.05% Tween 20


93

Figure 4.9 Standard curves of ENR in different diluents (PBS, 1% FG‐PBS, 1% FG‐PBS + 0.05%

Tween 20 and 1% FG‐PBS + 0.1% Tween 20). Each value represents the mean of triplicates

(n=3) with a standard deviation (SD) value of PBS, 1% FG‐PBS, 1% FG‐PBS + 0.05% Tween 20

and 1% FG‐PBS + 0.1% Tween 20 was 1.5, 3.4, 0.8 and 0.7, respectively. Statistical Analysis

was calculated using t‐test ( = 0.05).

4.3.4.2.2 Effects of organic solvents

Most FQ antibiotics are highly soluble in aqueous solutions either at high or low pHs, and

insoluble in many water miscible organic solvents. Amongst FQs, only ENR is soluble in

methanol. The solubility of an analyte is an aqueous solution can directly affect the assay

sensitivity. For this reason, various organic solvents at different concentrations were tested

for ENR solubility and their effects on the assay sensitivity. Colour development (Amax) and %

inhibition (IC50) reflecting the antibody binding to the immobilised antigen are presented in

Figures 4.10 to 4.17.

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

Inhibition (%)

ENR (µg L‐1)

PBS

1% FG‐PBS

1% FG‐PBS + 0.05% Tween 20


94

Figure 4.10 Effects of methanol (5% MeOH, 10% MeOH and 20% MeOH) on colour

development of the ENR1‐ELISA.Each value represents the mean of triplicates (n=3) with a

standard deviation (SD) value of PBS, 5% MeOH, 10% MeOH and 20% MeOH was 0.01, 0.01,

0.02 and 0.01, respectively. Statistical analysis was calculated using t‐test (α= 0.05)

Figure 4.11 Standard curves of ENR dissolved in different concentrations of methanol (5%

MeOH, 10% MeOH and 20% MeOH).Each value represents the mean of triplicates (n=3)

with a standard deviation (SD) value of PBS, 5% MeOH, 10% MeOH and 20% MeOH was 1.1,

0.9, 4.7 and 0.8, respectively. Statistical analysis was calculated using t‐test (α= 0.05)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

PBS

5% MeOH

10% MeOH

20% MeOH

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

PBS5% MeOH10% MeOH20% MeOH

95

Figure 4.12 Effects of acetonitrile (5% acetonitrile, 10% acetonitrile and 20% acetonitrile) on

colour development. Each value represents the mean of triplicates (n=3) with a standard

deviation (SD)value of PBS, 5% acetonitrile, 10% acetonitrile and 20% acetonitrile was 0.03,

0.03, 0.03 and 0.02, respectively. Statistical analysis was calculated using t‐test (α= 0.05).

Figure 4.13 Standard curves of ENR in different concentrations of acetonitrile (5%

acetonitrile, 10% acetonitrile and 20% acetonitrile). Each value represents the mean of

triplicates (n=3) with a standard deviation (SD) of PBS, 5% acetonitrile, 10% acetonitrile and

20% acetonitrile was 0.2, 12.4, 2.2 and 3.6, respectively. Statistical analysis was calculated

using t‐test (α= 0.05).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

PBS

Acetonitrile 5%

Acetonitrile 10%

Acetonitrile 20%

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

PBS

Acetonitrile 5%

Acetonitrile 10%

Acetonitrile 20%

96

Figure 4.14 Effects of acetone (5% acetone, 10% acetone and 20% acetone) on colour

development. Each value represents the mean of triplicates (n=3) with a standard

deviation (SD) of PBS, 5% acetone, 10% acetone and 20% acetone was 0.07, 0.03, 0.02 and

0.04, respectively. Statistical analysis was calculated using t‐test (α= 0.05).

Figure 4.15 Standard curves of ENR in different concentrations of acetone (5% acetone, 10%

acetone and 20% acetone). Each value represents the mean of triplicates (n=3) with a

standard deviation (SD) of PBS, 5% acetone, 10% acetone and 20% acetone was 2.2, 3.7,

1.8 and 5.1, respectively. Statistical analysis was calculated using t‐test (α= 0.05).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

PBS

Acetone 5%

Acetone 10%

Acetone 20%

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

PBS

Acetone 5%

Acetone 10%

Acetone 20%

97

Figure 4.16 Effects of ethanol (5% ethanol, 10% ethanol and 20% ethanol) on colour

development of the ENR1‐ELISA. Each value represents the mean of triplicates (n=3) with a

standard deviation (SD) of PBS, EtOH 5%, EtOH 10% and EtOH 20% was 0.03, 0.01, 0.01 and

0.03, respectively. Statistical analysis was calculated using t‐test (α= 0.05).

Figure 4.17 Standard curves of ENR in different concentrations of ethanol (5% ethanol, 10%

ethanol and 20% ethanol) for ENR1‐ELISA and each value represents the mean of triplicates

(n=3) with a standard deviation (SD) of PBS, EtOH 5%, EtOH 10% and EtOH 20% was 1.9, 4.1,

2.5 and 3.4, respectively. Statistical analysis was calculated using t‐test (α= 0.05).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

PBS

EtOH 5%

EtOH 10%

EtOH 20%

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

PBS

EtOH 5%

EtOH 10%

EtOH 20%

98

Table 4.6 Effects of water miscible organic solvents on the performance of the ENRELISA

Conc. of solvent (%v/v)

Methanol Acetonitrile Acetone Ethanol

Amax

± SD (n =3)

IC50 (µg L‐1) ±SD (n =3)

Amax

± SD (n =3)

IC50 (µg L‐1) ±SD (n =3)

Amax

± SD (n =3)

IC50 (µg L‐1) ±SD (n =3)

Amax

± SD (n =3)

IC50 (µg L‐1) ±SD (n =3)

0% 1.4±

0.01

12.8±

1.1

1.5±

0.03

11.0±

0.2

1.3±

0.07

11.1±

2.2

1.4±

0.03

14.7±

1.9

%CV (0%) 0.7 8.6 2.0 1.8 5.4 19.8 2.1 12.9

5% *1.4±

0.01

*13.3±

0.9

^1.0±

0.03

^26.3±

12.4

^0.9±

0.03

^107.8±

3.7

^0.9±

0.01

^79.7±

4.1

%CV (5%) 0.7 6.8 3.0 47.1 3.3 3.4 1.1 5.1

10% *1.6±

0.01

*17.0±

4.7

^1.1±

0.03

^19.2±

2.2

^1.1±

0.02

^126.5±

1.8

^1.0±

0.01

^104.7±

2.5

%CV

(10%) 0.6 27.6 2.7 11.5 1.8 1.4 1 3.4

20% ^1.9±

0.01

^22.4±

0.8

^1.3±

0.02

^18.9±

3.6

^1.1±

0.04

^157.1±

5.1

^1.2±

0.03

^128.9±

13.4

%CV

(20%) 0.5 3.6 1.5 19.0 3.6 3.2 2.5 10.4

Each value represents a mean of triplicates (n=3) with a standard deviation (SD). Statistical

analysis was calculated using t‐test (α = 0.05). *no significant difference with PBS.

^significant difference with PBS.

The effects of organic solvents (methanol, ethanol, acetone and acetonitrile between 5%

and 20% in PBS) on ELISA are shown in Table 4.7. Methanol and acetonitrile had little or no

effects on the sensitivity, compared to ethanol and acetonitrile. The IC50 values increased

gradually from 13.1 to 22.4 µg L‐1 as methanol concentration increased from 5 to 20%.In

contrast, the IC50 values decreased gradually from 26.3 to 18.9 µg L‐1 as acetonitrile

concentration increased from 5 to 20%. Meanwhile, the IC50 values increased dramatically

to more than ten‐folds when ethanol or acetone was present in the assay buffer. Methanol

at 5 and 10% did not significantly affect the colour development. For other solvents, the

Amax was affected considerably as their concentrations increased.

99

In summary, methanol up to 10% and acetonitrile up to 20% have no considerable

interference on the assay performance and are still able to maintain the same sensitivity.

Hence methanol up to 10 % was used routinely in the ENR‐1‐ELISA.

4.3.4.2.3 Effect of pH

Antibodies and enzymes are susceptible to extreme pHs. They generally have an optimum

pH range within which they function optimally. They are normally stable at a neutral pH

tolow alkaline conditions, ranging between pH 7 and 9. Acidic pH lower than pH 5 can

irreversibly inhibit the protein or enzyme activity (Hermanson, 1996). The pH of an

aqueous environment also influences ionic states of both the analytes and antibodies,

hence they may affect sensitivity and specificity of the assay (Sheng et al., 2009b). To

investigate the effects of pH on assay performance, standard curves of ENR prepared in

pH5.5, 6.5, 8.5 and 9.5 were compared with that of a curve generated using PBS at pH 7.4,

as a control.

Figure 4.18 illustrates the effects of pH on assay performance. There were no significant

changes in the IC50 values in a pH range of 6.5 and 8.5. This indicated that the assay was

stable in the pH range tested. At pHs 5.5 and 9.5, the IC50 values increased by 3‐ and 2‐folds,

respectively. With regard to the colour development, Amax gradually decreased at pHs

above and below 7.5.The optimum pH for this assay was atneutral (pH 7), hence, PBS (pH

7.4) solution was employed for further characterisation.

100

Figure 4.18 Effects of the pH on the ENR1‐ELISA. The circle indicates absorbance and the

triangle indicates IC50 values against pH and each value represents a mean of triplicates

(n=3) of pH 5.5, 6.5, 7.5, 8.5 and 9.5 with a standard deviation (SD) value was 4.5, 0.8, 0.1,

4.5 and 0.6, respectively. Statistical analysis was calculated using t‐test (α = 0.05).

4.3.5 Matrix Interferences

An ELISA is usually affected by the chemical, physical and biological components on the

matrices such as pigments, tannins, protein and fat (Lee and Kennedy, 2007). Matrix

interference of the samples may affect either assay sensitivity or the colour development

or both of these. There are a number of ways to remove or minimise the influence of

matrix interference on an ELISA. Common procedures for ELISA are extraction with water

miscible solvents, acidic or enzymatic hydrolysis, or simple dilution. Extraction of an

analyte from a sample matrix is influenced by many factors including solubility of an

analyte and interfering substances, diffusion rate of solvent into a sample matrix, partition

of the analyte between sample matrix and extraction solvent (mass transfer)and particle

size to which matrix is ground (Mitra, 2003). Solid samples are generally prepared by

grinding into finer size particles first, followed by liquid extraction using appropriate

solvents and then the extract is either concentrated or undergoing an additional

interference removal procedures (Ridgway et al., 2007). Moist or wet samples, such as

animal or fish tissues are generally prepared by mincing or homogenising. Liquid samples,

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

5.5 6.5 7.5 8.5 9.5

Absorban

ce (450 nm)

IC50(µg L‐1)

pH

101

such as syrups, soft drinks, milks or other liquid drinks can be prepared by a simple dilution

with aqueous solutions, extraction using water immiscible solvents to remove specific

matrix interference(e.g., fat in milk or pigment in vegetables), and addition of chelating

agents (e.g., interfering ions in soft drinks).

In this study solid samples (chicken liver and prawn) were homogenised using a

homogeniser and the homogenised sample was extracted three times with 50mM NaOH :

methanol : PBS = 1:9:90 in a shaking waterbath at 40oC for 15 min without further

treatment. Meanwhile, liquid samples (milk) were diluted with PBS (1:5, 1:10, and 1:20),

followed by heating in a waterbath at 80oC for 5 min and 30 min for skim milk and full

cream milk samples, respectively. The matrix effects were examined by running a serial

dilution of ENR in the extracts or diluted samples and comparing the results with those

obtained in PBS buffer as a control. The matrix interference was evaluated by comparing

the maximum absorbance (Amax) and the sensitivity (IC50) as the indicators of antibody‐

antigen binding and enzyme activity.

4.3.5.1 Effect of matrix in milk

Skim milk has much lower fat contents (0.3%) than full cream milk (3.3% to 5%). To

evaluate the effects of fat contents in various milk samples on the ELISA, the fat globules

were dispersed by preheating the sample in a waterbath at 80oC for 5 min. In this study,

three dilutions (1:5, 1:10 and 1:20) were examined to compare the effects on the colour

development (Amax) and % inhibition (IC50) (Figures 4.19 to 4.26).

102

Figure 4.19 Effects of skim milk liquid (SKL), diluted 1:5, 1:10 and 1:20 with PBS on colour


deviation (SD) value of PBS, diluted SKL 1:5, 1:10 and 1:20 with PBS was 0.01, 0.04, 0.01

and 0.04, respectively. Statistical analysis was calculates using t‐test (α = 0.05).

Figure 4.20 Standard curves of ENR dissolved in skim milk liquid (SKL), diluted 1:5, 1:10 and

1:20 with PBS. Each value represents the mean of triplicates (n=3) with standard deviation

(SD) value of PBS, diluted SKL 1:5, 1:10 and 1:20 with PBS was 0.8, 1.7, 1.2 and 0.6,

respectively. Statistical analysis was calculated using t‐test (α = 0.05).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

1 in 5

1 in 10

1 in 20

PBS

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

1 in 5

1 in 10

1 in 20

PBS

103

Figure 4.21 Effects of skim milk powder (SKP), diluted 1:5, 1:10 and 1:20 with PBS, on

colour development of the ENR1‐ELISA. Each value represents the mean of triplicates (n=3)

with standard deviation (SD) of PBS, diluted SKP 1:5, 1:10 and 1:20 with PBS was 0.01, 0.04,

0.01 and 0.04, respectively. Statistical analysis was calculated using t‐test (α = 0.05).

Figure 4.22 Standard curves of ENR dissolved in skim milk powder (SKP), 1:5, 1:10 and 1:20

with PBS. Each value represents the mean of triplicates (n=3) with a standard deviation (SD)

of PBS, diluted SKP 1:5, 1:10 and 1:20 with PBS was 0.7, 1.6, 1.2 and 0.6, respectively.

Statistical analysis was calculated using t‐test (α = 0.05).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

1 in 5

1 in 10

1 in 20

PBS

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibtion

ENR (µg L‐1)

1 in 5

1 in 10

1 in 20

PBS

104

Figure 4.23 Effects of full cream milk liquid (FCL), diluted 1:5, 1:10 and 1:20 with PBS on

colour development of the ENR1‐ELISA. Each value represents the mean of triplicates (n=3)

with a standard deviation (SD) of PBS, diluted FCL 1:5, 1:10 and 1:20 with PBS was 0.01,

0.04, 0.01 and 0.04, respectively. Statistical analysis was calculated using t‐test (α = 0.05).

Figure 4.24 Standard curves of ENR dissolved in full cream milk liquid (FCL), diluted 1:5,

1:10 and 1:20. Each value represents the mean of triplicates (n=3) with a standard

deviation (SD) of PBS, diluted FCL 1:5, 1:10 and 1:20 with PBS was 0.8, 1.7, 1.2 and 0.6,


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

1 in 5

1 in 10

1 in 20

PBS

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

1 in 51 in 101 in 20PBS

105

Figure 4.25 Effects of full cream milk powder (FCP), diluted 1:5, 1:10 and 1:20 with PBS on

colour development. Each value represents the mean of triplicates (n=3) with a standard

deviation (SD) of PBS, diluted FCP 1:5, 1:10 and 1:20 with PBS was 0.01, 0.04, 0.01 and 0.04,


Figure 4.26 Standard curves of ENR dissolved in full cream milk powder (FCP), diluted 1:5,

1:10 and 1:20 with PBS. Each value represents the mean of triplicates (n=3) with a standard

deviation (SD) of PBS, diluted FCP 1:5, 1:10 and 1:20 with PBS was 1.1, 1.2, 0.9, 1.3,


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

1 in 5

1 in 10

1 in 20

PBS

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

1 in 51 in 101 in 20PBS

106

Table 4.7 Effects of milk matrix on colour development (Amax) and assay sensitivity (IC50).

Dilution

factor

(in PBS)

*SKL SKP FCL FCP

Amax

± SD

(n=3)

IC50

(µg L‐1)

±SD (n=3)

Amax

± SD

(n=3)

IC50

(µg L‐1)

±SD

(n=3)

Amax

± SD

(n=3)

IC50

(µg L‐1)

±SD

(n=3)

Amax

± SD

(n=3)

IC50

(µg L‐1)

±SD

(n=3)

1:5 ^1.4±

0.04

^19.3±

1.7

^1.3±

0.04

^14.2±

1.6

^1.2±

0.04

^19.3±

1.7

^1.1±

0.04

^18.8±

1.2

%CV (1:5) 3.3 8.9 3.3 11.3 3.3 8.8 3.3 6.4

1:10 ^1.5±

0.01

*12.6±

1.2

*1.4±

0.01

*12.4±

1.2

*1.4±

0.01

*13.9±

1.2

*1.4±

0.01

*12.9±

0.9

%CV(1:10) 0.7 8.4 0.7 9.5 0.7 8.3 *0.7 6.9

1:20 *1.6±

0.04

*12.7±

0.6

*1.5±

0.04

*9.0±

1.5

*1.4±

0.04

*12.7±

0.6

*1.4±

0.04

*12.4±

1.3

%CV (1:20) 0.7 4.7 0.7 16.7 0.7 4.7 0.7 10.5

PBS 1.6±

0.01

11.7±

0.8

1.5±

0.01

10.3±

0.7

1.4±

0.01

11.7±

0.8

1.5±

0.01

10.7±

1.1

%CV (PBS) 0.7 6.4 0.7 7.3 0.7 6.8 0.7 10.3

*SKL= skim milk liquid; SKP= skim milk powder; FCL= full cream liquid; FCP= full cream

powder. Each value represents the mean of triplicates (n=3) with standard deviation (SD).

Statistical analysis was calculated using t‐test (α = 0.05).*no significant difference with PBS

(control). ^significant difference with PBS (control).

As shown in Table 4.8, when dilution of milk increased from 1:5 to 1:20, the absorbance

gradually approached that of PBS buffer, indicating that increasing dilution decreased

matrix effects. The Amax values of skim milk and full cream milk at different dilutions did not

superimpose, larger differences were seen at dilutions lower than 5‐folds. The 10‐ and 20‐

fold dilutions did not significantly change Amax. It was apparent from the IC50 values that

the milk matrix inhibited the antibody‐antigen interaction. More analytically acceptable

results were obtained when the sample was diluted by 10‐folds or more with PBS.

Furthermore, the 10 and 20‐fold dilutions did not considerably affect the assay sensitivity.

107

While dilution is a solution to matrix interference, it also reduces assay sensitivity by the

same degree. To improve the sensitivity for diluted milk but still maintain low interference

of antibody‐antigen interaction, pre‐treatment was increased to 30 min, then followed by

centrifugation for 10 min before diluting with PBS. The results suggest that a ten‐fold

dilution would provide acceptable results (Figure 4.27 to 4.30).

Figure 4.27 Effects of skim milk liquid (SKL) and skim milk powder (SKP), diluted 1:10 with

PBS, on colour development of the ENR1‐ELISA.Each value represents the mean of five

replicates (n=5) with a standard deviation (SD) value of PBS, diluted SKL and SKP 1:10 with

PBS was 0.1, 0.2 and 0.2, respectively. Statistical analysis was calculated using t‐test (α =

0.05).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

PBS

SKL

SKP

108

Figure 4.28 Standard curves of ENR in skim milk liquid (SKL) and skim milk powder (SKP),

diluted 1:10 with PBS. Each value represents the mean of five replicates (n=5) with a

standard deviation (SD) value of PBS, diluted SKL and SKP 1:10 with PBS was 1.2, 2.5 and

2.8, respectively. Statistical analysis was calculated using t‐test (α = 0.05).

Figure 4.29 Effect of full cream milk liquid (FCL) and full cream milk powder (FCP), diluted

1:10 with PBS, on colour development. Each value represents the mean of five replicates

(n=5) with a standard deviation (SD) of PBS, diluted SKL and SKP 1:10 with PBS was 0.1, 0.2

and 0.1, respectively. Statistical analysis was calculated using t‐test (α = 0.05).

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

PBS

SKL

SKP

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

PBS

FCL

FCP

109

Figure 4.30 Standard curves of ENR dissolved in full cream milk liquid (FCL) and full cream

milk powder (FCP), diluted 1:10 with PBS. Each value represents the mean of five replicates

(n=5) with a standard deviation (SD) of PBS, diluted SKL and SKP 1:10 with PBS was 1.6, 2.0

and 2.0, respectively. Statistical analysis was calculated using t‐test (α = 0.05).

Table 4.8 Matrix effects of pre‐treated milk, diluted 1:10 with PBS, on colour development

(Amax) and assay sensitivity (IC50)

Milk samples Skim milk ± SD (n=5) Full cream milk ± SD (n=5)

PBS SKL SKP PBS FCL FCP

Amax 1.6 ± 0.1 1.4 ± 0.2 1.4 ± 0.2 1.6 ± 0.1 1.5 ± 0.2 1.4 ± 0.1

%CV(Amax) 0.7 11.3 10.8 3.7 10.4 9.4

IC50 (µg L‐1) 10 ± 1.2 13 ± 2.5 14 ± 2.8 9 ± 1.6 12 ± 2.0 12 ± 2.0

%CV(IC50) 16.8 19.4 23.8 12.0 17.8 16.6

Each value represents the mean of five replicates (n=5) with standard deviation (SD).

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% inhibition

ENR (µg L‐1)

PBS

FCL

FCP

110

4.3.5.2 Effect of matrix in chicken liver and shrimp samples

FQ antibiotics are only soluble in certain organic solvents, hydro‐organic (a mixture of

organic solvent and aqueous acidic or basic solution), aqueous acidic or basic media.

Therefore, adding organic solvent or basic aqueous solution or a mixture of those solvents

to PBS would help to increase the solubility of FQ residues and subsequent extractability

during sample extraction.

To reduce matrix interference from chicken liver and prawn, both extraction and dilution

were employed. Briefly, chicken liver from a local butcher (Lv3) and local prawn (Pr1) were

extracted with a mixture of 50mM sodium hydroxide, methanol, PBS (1:9:90) and the

extracts were diluted in 1:20 with PBS. As shown in Table 4.10, a 20‐fold dilution increased

the sensitivity by approximately two‐fold for both chicken liver and prawn samples. It can

be seen from the IC50 values that the extracts of blank samples diluted in 1:20 with PBS still

showed interfering and inhibiting effects on antibody‐antigen binding. Moreover, the

extracts diluted in 1:20 with PBS did not yield curves that are super imposable on the

curves of PBS and extraction solvents (50 mM NaOH, methanol, PBS = 1:9:90) (Figures 4.31

to 4.34). Therefore, to improve the sensitivity and maximise antibody‐antigen interaction,

the dilution factor was increased to 1:50 with PBS.

111

Table 4.9 Effects of chicken liver (Lv2) and prawn (Pr1) samples, in a 20‐fold dilution with

PBS on colour development (Amax) and assay sensitivity (IC50).

Samples

Absorbance maximum (Amax) IC50 (µg L‐1)

PBS ±

SD (n=3)

*Extraction solvent (control)

± SD (n=3)

Extracted samples

± SD (n=3)

PBS ±

SD (n=3)

*Extraction solvent (control)

± SD (n=3)

Extracted samples

± SD (n=3)

Liver

(Coles, Lv2) 1.5 ± 0.03 *1.5 ± 0.04 ^1.8 ± 0.03 13.8 ± 0.8 *12.8 ± 0.9 ^22.8 ± 0.6

%CV (Lv2) 1.4 2.6 1.7 5.7 7.1 2.6

Prawn

(raw, local,

Pr1)

1.9± 0.02 *1.8 ± 0.01 ^2.2 ± 0.01 10.9 ± 0.7 *12.2 ± 3.1 ^27.1 ± 1.8

%CV (Pr1) 1.0 0.3 0.6 6.6 25.2 6.8

*Extraction solvent is 50 mM NaOH:MeOH:PBS=1:9:90.Each value represents the mean of

triplicates (n=3) with a standard deviation (SD).*no significant difference with PBS.

^significant difference with extraction solvent (control)

112

Figure 4.31 Effects of chicken liver (Coles, Lv2), diluted 1:20 with PBS, on colour


deviation (SD) value of PBS, extraction solvent and extracted liver was 0.03, 0.04 and 0.03,


Figure 4.32 Standard curves of ENR dissolved in chicken liver extract (Coles, Lv2).Each value

represents the mean of triplicates (n=3) with a standard deviation (SD) value of PBS,

extraction solvent and extracted liver was 0.8, 0.9 and 0.6, respectively. Statistical analysis

was calculated using t‐test (α = 0.05).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0.1 1 10 100

Absorban

ce

ENR (µg L‐1)

extracted liver (Lv1)

Extraction solvent

PBS

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

extracted liver (Lv1)

Extraction solvent

PBS

113

Figure 4.33 Effects of local prawn extract (Pr1) on colour development. Each value


extraction solvent and extracted liver was 0.02, 0.01 and 0.01, respectively. Statistical

analysis was calculated using t‐test (α = 0.05).

Figure 4.34 Standard curves of ENR dissolved in the local prawn extract (Pr1).Each value


extraction solvent and extracted liver was 0.7, 3.1 and 1.8, respectively. Statistical analysis

was calculated using t‐test (α = 0.05).

0.0

0.4

0.8

1.2

1.6

2.0

0.1 1 10 100 1000

Absorban

ce

ENR (µg L‐1)

Extraction solvent

Extracted prawn (Pr1)

PBS

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

Extraction solvent


PBS

114

Table 4.10 Effects of chicken liver and prawn extracts, on colour development (Amax) and

assay sensitivity (IC50).

Samples

Amax ± S.D. (n=5) IC50 (µg L‐1) ± S.D. (n=5)

PBS Extraction solvent

Extracted samples

PBS Extraction solvent

Extracted samples

Liver

(Organic, Lv1) 1.2 ± 0.2 1.2 ± 0.1 *1.2 ± 0.1 9 ± 2.2 11 ± 0.8 *11 ± 0.8

%CV (Lv1) 13.8 8.7 7.9 23.6 7.7 7.5

Liver

(Coles, Lv2) 1.2 ± 0.2 1.4 ± 0.2 *1.3 ± 0.2 11 ± 2.6 13 ± 2.9 *13 ± 2.0

%CV (Lv2) 17.9 11.8 12.7 24.6 21.5 15.4

Liver

(Butcher, Lv3) 1.2 ± 0.2 1.4 ± 0.2 *1.4 ± 0.2 13 ± 5.4 14 ± 4.7 *15 ± 3.5

%CV (Lv3) 16.6 12.0 11.8 42.2 31.5 24.7

Prawn

(raw, local, Pr1) 1.9 ± 0.2 1.8 ± 0.2 *1.8 ± 0.2 12 ± 6.3 14 ± 2.7 *15 ± 3.6

%CV (Pr1) 12.7 9.3 9.0 53.4 18.8 23.9

Prawn

(cooked, imported‐

Thailand, Pr2)

1.5 ± 0.1 1.5 ± 0.1 *1.5 ± 0.1 9 ± 2.2 14 ± 1.6 *15 ± 2.5

%CV (Pr2) 2.5 0.6 1.8 23.6 13.8 14.8

Prawn

(peeled off, imported‐

Malaysia, Pr3)

1.5 ± 0.1 1.5 ± 0.1 *1.5 ± 0.1 12 ± 4.4 14 ± 1.3 *16 ± 2.3

%CV (Pr3) 2.4 0.6 1.1 37.6 9.2 14.0

Extracts were prepared by diluting 1:50 with PBS. Each value represents the mean of

triplicates (n=3) with a standard deviation (SD). Statistical analysis was calculated using t‐

test (α = 0.05). *no significant difference with PBS and control.

115

As shown in Table 4.11, an improved sample extraction by increasing the dilution factor to

50‐fold showed apparent removal of matrix effects in all solid samples (chicken liver and

prawn), resulting in a better assay sensitivity of about 13 µg L‐1. The IC50 values of 1:50

dilution decreased by approximately two‐fold compared to that obtained in 1:20 dilution. A

50‐fold dilution yielded the IC50 values between 11 ± 0.8 µg L‐1 and 13 ± 5.4 µg L‐1in the

extraction solvents and 11 ± 0.8 µg L‐1 and 16 ± 2.3 µg L‐1 in liver and prawn sample extracts,

respectively. Also, there was no significant difference in the IC50 values obtained from the

extraction solvent and food sample extracts compared to that of PBS buffer (matrix free).

Evidently, a 50‐fold dilution was adequate to remove interferences from liver and prawn

matrices. Therefore, a mixture of 50mM sodium hydroxide, methanol, PBS (1:9:90) as a

solvent extraction followed by a 50‐fold dilution with PBS were then adopted in the spike

and recovery studies (Figures 4.35 to 4.46).

116

Figure 4.35 Effects of organic chicken liver (Lv1), diluted 1:50 with PBS, on colour

development. Each value represents the mean of five replicates (n=5) with a standard

deviation (SD) value of PBS, extraction solvent, and extracted organic chicken liver (Lv1)

was 0.2, 0.1 and 0.2, respectively. Statistical analysis was calculated using t‐test (α = 0.05)

Figure 4.36 Standard curves of ENR dissolved inorganic chicken liver extract (Lv1).Each

value represents the mean of replicates (n=5) with a standard deviation (SD) value of PBS,

extraction solvent, and extracted organic chicken liver (Lv1) was 2.2, 0.8 and 0.8,

respectively. Statistical analysis was calculated using t‐test (α = 0.05)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.1 1 10 100 1000

Ab

sorb

ance

(45

0 n

m)

ENR (µg L-1)

Extraction solvent

Extracted liver (Lv1)

PBS

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

Extraction solvent


PBS

117

Figure 4.37 Effects of chicken liver extract (Lv2), diluted 1:50 with PBS, on colour


deviation (SD) of PBS, extraction solvent, and extracted chicken liver (Lv2) was 0.2, 0.2 and

0.2, respectively. Statistical analysis was calculated using t‐test (α = 0.05)

Figure 4.38 Standard curves of ENR dissolved in chicken liver extract (Lv2), diluted 1:50

with PBS. Each value represents the mean of five replicates (n=5) with a standard deviation

(SD) of PBS, extraction solvent, and extracted chicken liver (Lv2) was 2.6, 2.9 and 2.0,


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

Extraction solvent


PBS

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

Extraction solvent


PBS

118

Figure 4.39 Effects of chicken liver extract (Lv3), diluted 1:50 with PBS, on colour


deviation (SD) of PBS, extraction solvent, and extracted chicken liver (Lv2) was 0.2, 0.2 and

0.2, respectively. Statistical analysis was calculated using t‐test (α = 0.05)

Figure 4.40 Standard curves of ENR dissolved in a chicken liver extract (Lv3).Each value

represents the mean of five replicates (n=5) with a standard deviation (SD) of PBS,

extraction solvent, and extracted chicken liver (Lv3) was 5.4, 4.7 and 3.5, respectively.

Statistical analysis was calculated using t‐test (α = 0.05)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

Extraction solvent


PBS

0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

Extraction solvent


PBS

119

Figure 4.41 Effects of local prawn extract (Pr1), diluted 1:50 with PBS, on colour


deviation (SD) of PBS, extraction solvent, and extracted prawn (Pr1) was 0.2, 0.2 and 0.2,


Figure 4.42 Standard curves of ENR in local prawn extract (Pr1).Each value represents the

mean of five replicates (n=5) with a standard deviation (SD) of PBS, extraction solvent, and

extracted local prawn (Pr1) was 6.3, 2.7 and 3.6, respectively. Statistical analysis was

calculated using t‐test (α = 0.05).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

PBS

Extraction Solvent


0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

PBS

Extraction Solvent


Figur

colou

stand

0.1 a

Figur

supe

devia

respe

re 4.43 Effe

ur develop

dard deviat

and 0.1 resp

re 4.44 Sta

ermarket (P

ation (SD)P

ectively. Sta

cts of praw

ment. Each

tion (SD) PB

pectively. St

ndard curv

r2).Each va

PBS, extract

atistical ana

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1

Absorban

ce (450 nm)

n from Cole

h value rep

BS, extractio

tatistical ana

es of ENR

lue represe

tion solvent

alysis was ca

1

120

es superma

presents th

on solvent,

alysis was c

in an extra

ents the me

t, and extr

alculated us

10ENR (µg

rket (Pr2), e

he mean o

, and extrac

calculated u

act from th

ean of five r

racted praw

sing t‐test (α

100g L‐1)

PBS

Extraction

Extracted

extract dilut

of five repl

cted local p

sing t‐test (

e prawn pu

replicates (n

wn (Pr2) w

α = 0.05).

1000

n Solvent

prawn (Pr2)

ted 1:50 wi

licates (n=5

prawn (Pr2)

(α = 0.05).

urchased fr

n=5) with a

as 2.2, 1.6

th PBS on

5) with a

) was 0.1,

rom Coles

standard

and 2.5,

121

Figure 4.45 Effects of extract from the prawn purchased from a local butcher (Pr3) on

colour development. Each value represents the mean of five replicates (n=5) with a

standard deviation (SD) PBS, extraction solvent, and extracted prawn (Pr3) was 0.1, 0.1 and

0.1, respectively. Statistical analysis was calculated using t‐test (α = 0.05).

Figure 4.46 Standard curves of ENR in an extract from the prawn purchased from a local

butcher (Pr3). Each value represents the mean of five replicates (n=5) with a standard

deviation (SD) PBS, extraction solvent, and extracted prawn (Pr3) was 4.4, 1.3 and 2.3,


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0.1 1 10 100 1000

Absorban

ce (450 nm)

ENR (µg L‐1)

PBS

Extraction Solvent


0

10

20

30

40

50

60

70

80

90

100

0.1 1 10 100 1000

% In

hibition

ENR (µg L‐1)

PBS

Extraction Solvent


122

4.3.6 Recovery studies

The MRLs for FQ residues in food are in the range of 50 to 1000 µg kg‐1 (Hernandez‐

Arteseros et al., 2002). For ENR and its metabolites in particular, the MRLs are between

100 and 300 µg kg‐1in edible animal tissues, muscle, seafood, and milk (Hernandez‐

Arteseros et al., 2002). Three food samples (milk, chicken liver and prawn) were chosen to

evaluate anayte recovery by the ELISA. ENR was spiked in PBS buffer or extraction solvent

and in milk samples (skim and full cream milk) each at four concentrations (50, 100, 200

and 500 µg L‐1).Chicken liver and prawn samples were spiked at 250, 500, 1000 and 2500

µg L‐1 (Table 4.1). Spiking ENR in the tested samples were based on their respective MRL

ideally covering a range that was ten times lower and higher than the MRL values(10 and

3000 µg kg‐1). The final concentrations of ENR in samples after the dilution (a 10‐fold in

milk and a 50‐fold in chicken liver and prawn samples) were5, 10, 20 and 50 µg L‐1.The

spiked samples were analysed by ENR1‐ELISA using AbENR1‐KLH and ENR1‐OA

combination.

123

Table 4.11 %Recoveries ENR spiked in milk as detected by ELISA.

Samples (liquid)

Spiked concentration

(µg L‐1)

Detected concentration (µg L‐1) ± SD (n=3)

Recovery (%) ± SD (n=3)

in PBS in diluted samples

in PBS in diluted samples

Skim milk (liquid) (SKL)

50

100

200

500

54.4 ± 4.2

122.9 ± 8.2

201.8 ± 8.8

445.1 ± 30.6

59.7 ± 4.2

125.1 ± 8.3

204.5 ± 8.8

462.7 ± 33.9

109 ± 8

123 ± 8

101 ± 4

89 ± 6

119 ± 8

125 ± 8

102 ± 4

93 ± 7

Skim milk powder

(SKP)

50

100

200

500

55.9 ± 3.5

130.4 ± 7.3

225.6 ± 9.4

439.6 ± 26.7

52.9 ± 2.9

124.0 ± 7.2

219.0 ± 9.5

494.8 ± 41.2

112 ± 7

130 ± 7

113 ± 5

88 ± 5

106 ± 6

124 ± 7

110 ± 5

99 ± 8

Full cream milk (liquid)

(FCL)

50

100

200

500

32.7 ± 2.0

79.8 ± 10.2

143.0 ± 8.0

541.2 ± 26.0

34.0 ± 1.8

71.4 ± 7.6

137.7 ± 8.6

559.6 ± 25.4

65 ± 4

80 ± 10

72 ± 4

108 ± 5

68 ± 4

71 ± 8

69 ± 4

112 ± 5

Full cream milk(powder)

(FCP)

50

100

200

500

39.7 ± 4.3

71.0 ± 5.2

140.4 ± 3.6

594.9 ± 20.5

35.3 ± 3.6

60.7 ± 4.2

130.8 ± 3.4

577.4 ± 20.5

79 ± 9

71 ± 11

70 ± 5

119 ± 8

71 ± 7

61 ± 9

65 ± 4

116 ± 8

Each value represents the mean of triplicates (n=3) with a standard deviation (SD).

Statistical analysis was calculated using t‐test (α = 0.05)

As can been seen on Table 4.12, the % recovery by the ELISA ranged between 89 (± 6) and

119 (± 8)% for skim milk liquid, 65 (± 4)% and 130 (± 7)% for skim milk powder, 69 (± 4)%

and 112 (± 5)% for full cream milk, 61 (± 9)% and 116 (± 8)% for full cream liquid. While

the % recovery of ENR by the ENR1‐ELISA was acceptable, generally, milk with low fat

contents will relatively higher recovery. For the later samples, the recovery rate was

dependent of concentration. That is, lower recovery was observed at lower concentrations

124

of ENR. Overall, good recoveries of ENR in milk samples were obtained. It can therefore be

concluded that the optimised sample preparation procedure with increasing preheating

time and centrifuging the sample to remove large particulates prior to further diluting with

PBS minimised significant matrix interference from milk.

Table 4.12% Recoveries of ENR spiked in chicken liver as detected by ELISA.

Solid samples (chicken liver)

Spiked conc. (µg L‐1)

Detected concentration (µg L‐1) ± SD (n=3)

Recovery (%) ± SD (n=3)

ENR spiked in extraction solvent

ENR spiked in extracted sample

ENR spiked in extraction solvent

ENR spiked in extracted sample

Chicken liver

(Organic,

Lv1)

250

500

1000

2500

147.7 ± 14.5

431.8 ± 40.0

683.8 ± 49.4

1553.2 ± 109.9

155.0 ± 15.1

541.9 ± 48.9

764.0 ± 54.4

1759.0 ± 128.9

59 ± 6

86 ± 8

68 ± 5

62 ± 4

62 ± 6

108 ± 10

76 ± 5

70 ± 5

Chicken liver

(Coles,

Lv2)

250

500

1000

2500

290 ± 23.1

678.8 ± 28.1

760.6 ± 44.1

1736.3 ± 134.2

257.5 ± 18.4

571.6 ± 28.6

690.7 ± 42.6

1599.2 ± 119.1

116 ± 9

136 ± 6

76 ± 4

70 ± 5

103 ± 7

114 ± 6

69 ± 4

64 ± 5

Chicken liver

(Butcher,

Lv3)

250

500

1000

2500

288.2 ± 14.6

370.3 ± 27.6

1173.6 ± 107.8

1847.5 ± 154.3

216.4 ± 17.9

321.9 ± 36.3

1057.8 ± 77.1

1521.0 ± 245.4

115 ± 9

74 ± 9

117 ± 9

74 ± 6

87 ± 10

64 ± 12

106 ± 7

61 ± 5


analysis was calculated using t‐test (α = 0.05)

125

Table 4.13% Recoveries of ENR spiked in prawn as detected by ELISA

Solid

samples

(prawn)

Spiked

conc.

(µg L‐1)

Detected concentration

(µg L‐1) ± SD (n=3)

Recovery (%)

± SD (n=3)

ENR spiked in

extraction

solvent

ENR spiked in

extracted

sample

ENR spiked

in extraction

solvent

ENR spiked

in extracted

sample

Prawn

(raw, local,

Pr1)

250

500

1000

2500

151.4 ± 9.6

300.3 ± 23.6

1069.1 ± 91.9

1963.5 ± 136.6

238.0 ± 12.8

428.7 ± 27.5

1200.1 ± 83.7

2116.7 ± 140.3

61 ± 4

60 ± 5

107 ± 9

79 ± 5

95 ± 5

86 ± 6

120 ± 8

85 ± 6

Prawn

(cooked,

imported‐

Thailand,

Pr2)

250

500

1000

2500

179.2 ± 4.6

421.9 ± 26.3

742.5 ± 68.1

1738.5 ± 57.7

168.0 ± 4.0

355 ± 19.7

637.9 ± 67.4

1647.1 ± 51.3

72 ± 2

84 ± 5

74 ± 7

70 ± 2

67 ± 2

72 ± 4

64 ± 7

66 ± 2

Prawn

(peeled off,

imported‐

Malaysia,

Pr3)

250

500

1000

2500

166.7 ± 15.4

330.8 ± 12.9

643.7 ± 30.1

1693.5 ± 158.0

185.1 ± 20.2

416.9 ± 19.4

812.2 ± 33.9

2106.3 ± 189.9

67 ± 6

66 ± 3

64 ± 3

68 ± 6

74 ± 8

83 ± 4

81 ± 3

84 ± 8


analysis was calculated using t‐test (α = 0.05)

As shown on Table 4.13, the % recovery ranged from 59 (± 6) to 108 (± 10)% for organic

chicken liver, 64 (± 5) to 136 (± 6)% for regular chicken liver from Coles supermarket, 61 (±

5) to117 (± 9)% for the regular chicken liver from a local butcher shop, 60 (± 5) to 120 (± 8)%

for the local prawn, 66 (± 2) to 84 (± 5)% for the imported prawn (Thailand), 64 (± 3) to 84

(± 8)% for the imported prawn (Malaysia). In general, the % recovery of prawn was lower

than those of milk. Differences were observed in % recovery between raw and cooked

prawn, suggesting ENR may be more tightly absorbed to the denatured prawn protein than

the unprocessed protein. Overall, the recoveries obtained from chicken liver and prawn

126

samples, although slightly lower than the generally accepted range of 80‐120% for ELISA,

were acceptable considering extraction of (incurred) ENR residue from prawn have been

known to be challenging, resulting in < 80% recovery even for the instration techniques.

Further refinement of the sample preparation and improvement of recovery rate would be

desirable.

4.3.7 Linear regression of spiking and recoveries

A linear regression of spike and recovery data of ENR spiked in PBS buffer and diluted

samples (for milks), and in extraction solvent and extracted samples (for chicken liver and

prawn) was evaluated, (Figure 4.47 to 4.50). The linearity or coefficient of correlation (R2)

values obtained in liquid samples (milks) were 0.985 ± 0.01, %CV = 0.8% (in PBS buffer),

and 0.988 ± 0.01, %CV = 1.1% (in diluted samples), and in solid samples (chicken liver and

prawn) were 0.973 ± 0.03, %CV = 3.0% (in extraction solvent) and 0.968 ± 0.04, %CV = 4.6%

(in extracted samples).

Figure 4.47 Correlation between the levels of ENR spiking in skim milk liquid (SKL), skim

milk powder (SKP), full cream milk liquid (SKL), full cream milk powder (SKP) and estimates

by the ENR1‐ELISA. Average values (µg L‐1) of spiking and spiking level (µg L‐1) from in PBS

buffer.

0

100

200

300

400

500

0 100 200 300 400 500

Spiked (µg L‐1)

ENR‐ELISA (µg L‐1)

SKL (y = 0.843x + 26.75; R2 = 0.995)

SKP (y = 0.820x + 38.72; R2 = 0.984)

FCL (y = 1.143x ‐ 43.78; R2 = 0.985)

FCP (y = 1.270x ‐ 58.45; R2 = 0.976)

127

Figure 4.48 Correlation between the levels of ENR spiking in skim milk liquid (SKL), skim

milk powder (SKP), full cream milk liquid (SKL), full cream milk powder (SKP) and estimates

by the ENR1‐ELISA. Average values (µg L‐1) of spiking and spiking level (µg L‐1) from in

diluted samples.

Figure 4.49 Correlation between the levels of ENR spiking in organic chicken liver (Lv1),

chicken liver form Coles (Lv2), chicken liver from butcher (Lv3), local prawn (Pr1), Thai

prawn (Pr2) and Malaysian prawn (Pr3) and estimates by the ENR1‐ELISA. Average values

(µg L‐1) of spiking and spiking level (µg L‐1) from in extraction solvent.

0

100

200

300

400

500

0 100 200 300 400 500

Spiked (µg L‐1)


SKL (y = 0.875x + 27.14; R2 = 0.997)

SKP (y = 0.9600x + 18.72; R2 = 0.996)

FCL (y = 1.143x ‐ 53.4; R2 = 0.981)

FCP (y = 1.244x ‐ 63.50; R2 = 0.976)

150

650

1150

1650

2150

2650

150 650 1150 1650 2150 2650

Spiked (µg L‐1)

ENR‐1 ELISA (µg L‐1)

Lv1 (y=0.809+10.89; R2=0.959)

Lv2 (y=0.680x+47.28; R2=0.998)

Lv3 (y=0.687x‐28.76; R2=0.999)

Pr1 (y=0.600x+62.28; R2=0.991)

Pr2 (y=0.600x+228.6; R2=0.968)

Pr3 (y=0.703x+174.2; R2=0.924)

128

Figure 4.50 Correlation between the levels of ENR spiking in organic chicken liver (Lv1),

chicken liver form Coles (Lv2), chicken liver from butcher (Lv3), local prawn (Pr1), Thai

prawn (Pr2) and Malaysian prawn (Pr3) and estimates by the ENR1‐ELISA. Average values

(µg L‐1) of spiking and spiking level (µg L‐1) from in extracted samples.

4.4 Conclusion

This chapter presented the development of an indirect competitive ELISA method for

quantification of ENR in milk, chicken liver and prawn. The ENR specific polyclonal

antibodies were raised against the conjugates of the ENR hapten, to KLH, as a carrier

protein, using the carbodiimide‐NHS mediated coupling reaction. The ENR‐1 ELISA

exhibited an IC50 value of 11.8 µg L‐1, a LOD of 2.4µg L‐1 and a LOQ of 8.0 µg L‐1, evidently

exhibiting high sensitivity of the assay.

The homologous system generated higher sensitivity compared to the heterologous

systems in this study. The hapten homologous and linkage heterologous (i.e., using ENR2‐

OA as a competing antigen) system exhibited poorer sensitivity and displayed very low

colour development. The Ab‐ENR1 was highly specific for ENR and no apparent cross‐

reactivity that is more than 0.1% was detected despite their close structural similarity to

ENR.

150

650

1150

1650

2150

150 650 1150 1650 2150 2650

Spiked (µg L‐1)


Lv1 (y = 0.674x + 89.26; R2 = 0.983)

Lv2 (y = 0.562x + 186.4 ; R2 = 0.982)

Lv3 (y = 0.579x + 163.5; R2 = 0.882)

Pr1 (y = 0.829x + 114.5; R2 = 0.960)

Pr2 (y = 0.652x + 9.641; R2 = 0.999)

Pr3 (y = 0.850x ‐ 24.97; R2 = 0.999)

129

The effects of surfactants (Tween 20), organic solvent (methanol, ethanol, acetonitrile and

acetone) and pH conditions (5.5‐9.5) were evaluated to optimize assay performance.

Tween 20 affected colour development, but it did not affect the assay sensitivity. Of the

tested solvents, only methanol gave no or very low interference on the assay performance.

Up to 10% methanol could be used without significantly affecting the assay sensitivity. No

significant changes in the IC50 values were observed for pH between 6.5 and 8.5, however,

a slight difference in colour development was found in those pH values. Hence, PBS with

pH 7.4 was employed for the ENR1‐ELISA.

The matrix interference of the developed ENR ELISA were studied by running calibration

curves prepared in three different samples matrices, namely milk, chicken liver and prawn.

Using the optimized pre‐treatment of milk followed by a 10‐fold dilution, the IC50 values

and colour development were similar to that of matrix free solution. The extraction of

chicken liver and prawn samples were optimized by using a mixture of extraction solvents

(50mMNaOH 0.5%, MeOH, PBS; 1:9:90) with a 50‐fold dilution in PBS. Recovery values for

milk, chicken liver and prawn were between 59 (± 6) and 136 (± 6)%. Therefore, it can be

concluded that the newly developed assay is suitable for the analysis of ENR in milk,

chicken liver and prawn samples.

130

CHAPTER 5. CONCLUSIONS and RECOMMENDATIONS

5.1 Conclusions

Fluoroquinolones (FQs) are one of the synthetic antibacterial groups administered as

medicine to treat and prevent various infectious diseases for both humans and animals

(Brown, 1996). Due to increasing concerns of FQ residues entering the food chain and

contributing to bacterial resistance that might affect human health, regulatory authorities

in many countries set up the maximum residue limits (MRLs) for FQs. The MRLs of FQs are

set in the range between 30 and 1500 µg kg‐1 according to different food resources (i.e.

animal edible tissue, meat, fish and milk) and authorities (i.e. U.S. FDA, FAO/WHO/JECFA,

EU, Canada, China and Japan) ( Lu et al., 2006, Zhao et al., 2007,Huang et al., 2010,).

Indonesia has had adopted and followed the MRLs for FQs and their metabolites at a range

from 100‐300 µg kg‐1 in any type of animal origin foods that have been set up by FAO/WHO

(JECFA) (Hernandez‐Arteseros et al., 2002). FQ antibiotics are recently becoming more

frequently used in livestock and aquaculture farms in Indonesia. According to the sample

entry data of pharmaceutical products from the National Veterinary Drug Assay Laboratory,

the Ministry of Agriculture, Gunungsindur‐Bogor, West Java Province, Indonesia, FQs

totalled 30% of pharmaceutical products used between 2004 and 2007. ENR has the largest

number of brand names amongst single active ingredient products, and about 54 brand

names of ENR being distributed in the Indonesian market. It is important for Indonesia to

evaluate the adequacy of MRLs for FQs that were adopted from FAO/WHO (JECFA).

Chemical instrumental methods, such as HPLC and LC‐MS have been widely used for

screening of FQ residues because of their accuracy and sensitivity. However, these

methods are time consuming, expensive, and they require highly trained staff. An

immunochemical assay is an alternative method that is fast and can be just as sensitive as

those instrumental methods, for routine screening as well as quantifying of FQs. As

immunochemical assay provides low operating costs, this would be beneficial for

laboratories which could not afford such expensive instrumentations, particularly in

developing countries, such as Indonesia, for routine screening or even quantification of FQ

residues in animal and marine derived products,. Moreover, increasing routine monitoring

131

of FQs residues of food products in Indonesia will ensure safer and healthier food and help

to increase its trade capacity.

This research project, therefore, aims to improve the sensitivity and specificity of

antibodies for the detection FQ residues in marine and animal‐derived products through

novel hapten design and synthesis. A series of novel ENR, ciprofloxacin and norfloxacin

hapten syntheses haptens were carried out. The haptens were designed and synthesised

based on the attachment of linkers on carboxylic or piperazinyl groups. The ENR hapten

was synthesised by attaching a tert‐butyl linker on the carboxylic group of ENR. The

piperazinyl group of ciprofloxacin and norfloxacin were attached with the linker containing

a4‐bromobutane NHS ester and a bromocrotyl NHS ester, respectively.

On the immunochemical method development, polyclonal antibodies raised against ENR

hapten‐KLH immunogen showed specific recognition to ENR without significant cross‐

reactivity to seven FQs structurally related compounds (danofloxacin, enofloxacin,

sarafloxacin, perfloxacin, nalidixic acid, ciprofloxacin and norfloxacin). The ELISAs had an

IC50 value of 11.7 µg L‐1 ± 1.7 and the limit of detection (LOD) value of 2.4 µg L‐1 ± 0.4.

The effects of surfactants (Tween 20), organic solvent (methanol, ethanol, acetonitrile and

acetone) and pH conditions (5.5‐9.5) were evaluated to optimize assay performance.

Tween 20 affected considerably on colour development, but it did not affect the assay

sensitivity. Up to 10% of methanol can be used without significantly affected with the assay

sensitivity. No significant changes in the IC50 values were observed for pH 6.5 to 8.5,

although a slightly different in colour development was found in pH below and above 7.4.

The sample preparation techniques were also optimized for milk, chicken liver and prawn,

yielding recoveries between 59 ± 6 and 136 ± 6%. ENR is known to bind to protein and it is

speculated that recovery rate being affected probably due to binding of ENR residues to

matrix proteins. This suggests further refinement of the sample preparation to improve the

recovery of ENR residues from chicken liver and prawn samples.

As shown in this thesis, the assay was able to generate highly specific assay for the

detection of a single FQ compound. This ELISA can be used for the routine screening and

quantitative analyses for ENR residues in animal and marine derived products.

132

5.2. Recommendations

Further validation of this assay with conventional instrumentation methods such as HPLC

or LC‐MS is needed as a future work. While immunoassays as demonstrated in this thesis is

simple, fast and sensitive, development of label‐free immunosensors using the existing

antibodies for detection of FQ residues in foods would provide further benefits of

immunodiagnostics technology.

Regards to Indonesia, the extend of FQ residues in animal and seafood samples is not

known and surveys of FQ residues in such samples from traditional markets are urgently

needed for adequately assess the potential risks pose on human health. The outcomes of

such studies may urge Indonesian regulatory agents to re‐enforce their regulations in order

to project the general public.

133

REFERENCES

Anadon, A., Martinez‐Larranaga, M. R., Diaz, M. J., Bringas, P., Martinez, M. A., Fernandez‐Cruz, M. L., Fernandez, M. C. & Fernandez, R. 1995. Pharmacokinetics and Residues Of ENR In Chickens. American Journal of Veterinary Research, 56, 501‐506.

Angulo, F. J., Johnson, K. R., Tauxe, R. V. & Cohen, M. L. 2000. Origins And Consequences Of Antimicrobial‐Resistant Nontyphoidal Salmonella: Implications for the Use of Fluoroquinolones in Food Animals. Microbial Drug Resistance, 6, 77‐83.

Ball, P. 2000. Quinolone Generations: Natural History Or Natural Selection? Journal of Antimicrobial Chemotherapy, 46, 17‐24.

Beier, R. C., Stanker, L. H. & 1996, I. F. R. A., American Chemical Society, Washington DC., USA. 1996. Immunoassays for Residue Analysis,. American Chemical Society.

Beneš, J. 2005. Review and Categorization Of Quinolone Antibiotics. Přehled A Rozdělení Chinolonových Antibiotik, 11, 4‐14.

Bertino, J. & Fish, D. 2000. The Safety Profile of The Fluoroquinolones. Clinical Therapeutics, 22, 798‐817.

Blondeau, J. M. 2004. Fluoroquinolones: Mechanis of Action, Classification, and Development of Resistance. Survey of Ophthalmology, 49, 73‐78.

Bogialli, S., D'ascenzo, G., Corcia, A. D., Lagana, A. & Nicolardi, S. 2008. A Simple and Rapid Assay Based on Hot Water Extraction and Liquid Chromatography‐Tandem Mass Spectrometry for Monitoring Quinolone Residue in Bovine Milk. Food Chemistry 180, 354‐360.

Brás Gomes, F. B. M., Riedstra, S. & Ferreira, J. P. M. 2010. Development Of an Immunoassay for Ciprofloxacin Based on Phage‐Displayed Antibody Fragments. Journal of Immunological Methods, 358, 17‐22.

Brown, S. A. 1996. Fluoroquinolones in Animal Health. Journal of Veterinary Pharmacological Therapy, 19.

Bucknall, S., Silverlight, J., Coldham, N., Thorne, L. & Jackman, R. 2003. Antibodies to the Quinolones and Fluoroquinolones for the Development of Generic and Specific Immunoassays for Detection of these Residues in Animal Products. Journal of Food Additives and Contaminants 20, 221‐228.

Burkin, M. A. 2008. Enzyme‐Linked Immunosorbent Assays Of Fluoroquinolones with Selective and Group Specificities. Journal of Food and Agricultural Immunology, 19, 131‐140.

Cao, L., Kong, D., Sui, J., Jiang, T., Li, Z., Ma, L. & Lin, H. 2009. Broad‐Specific Antibodies for A Generic Immunoassay Of Quinolone: Development of A Molecular Model for Selection of Haptens Based on Molecular Field‐Overlapping Journal of Analytical Chemistry, 81.

Cao, L., Lin, H. & Mirsky, V. M. 2007. Surface Plasmon Resonance Biosensor for ENR Based On Deoxyribonucleic Acid. Journal of Analytica Chimica Acta, 589, 1‐5.

Cao, L., Sui, J., Kong, D., Li, Z. & Lin, H. 2011. Generic Immunoassay of Quinolones: Production and Characterization Of Anti‐Pefloxacin Antibodies as Broad Selective Receptors. Journal of Food Analytical Methods, Doi: 10.1007/S12161‐011‐9196‐2.

Carlucci, G. 1998. Analysis of Fluoroquinolones in Biological Fluids By High Performance Liquid Chromatography. Journal of Chromatography, 812, 343‐367.

134

Chen, J., Xu, F., Jiang, H., Hou, Y., Rao, Q., Guo, P. & Ding, S. 2009a. A Novel Quantum Dot‐Based Fluoroimmunoassay Method for Detection Of ENR Residue in Chicken Muscle Tissue. Food Chemistry, 113, 1197‐1201.

Chen, J., Xu. F., Jiang, H., Hou, Y., Rao, Q., Guo, P. & Ding, S. 2009b. A Novel Quantum Dot‐Based Fuoroimmunoassay Method for Detection Of Enrofloxacin Residue in Chicken Muscle Tissue, Journal of Food Chemistry, 113, 1197‐1201.

Choma, I., Grenda, D., Malinowska, I. & Suprynowicz, Z. 1999. Determination of Flumequine and Doxycycline in Milk By A Simple Thin‐Layer Chromatographic Method. Journal of Chromatography B: Biomedical Sciences And Applications, 734, 7‐14.

Choma, I. M., Choma, A., Komaniecka, I., Pilorz, K. & Staszezuk, K. 2004. Semiquantitative Estimation of ENR And Ciprofloxacin by Thin‐Layer Chromatography ‐ Direct Bioautography. Journal of Liquid Chromatography and Related Technologies, 27, 2071‐2085.

Choma, I. M., Choma, A. & Staszczuk, K. 2002. Determination of Flumequine in Milk by Thin‐Layer Chromatography‐Bioautography. Journal of Liquid Chromatography & Related Technologies, 25, 1579 ‐ 1587.

Cinquina, A. L., Roberti, P., Giannetti, L., Longo, F., Draisci, R., Fagiolo, A. & Brizioli, N. R. 2003. Determination of ENR and Its Metabolite Ciprofloxacin in Goat Milk by High‐Performance Liquid Chromatography with Diode‐Array Detection: Optimization and Validation. Journal of Chromatography A, 987, 221‐226.

Coillie, E. V., Block, J. D. & Reybroeck, W. 2004. Development of An Indirect Competitive Elisa for Flumequine Residues in Raw Milk Using Chicken Egg Yolk Antibodies. Journal of Agricultural and Food Chemistry, 52, 4975‐4978.

Crowther, J. R. 2001. The Elisa Guidebook. Deshpande, S. S. 1996. Enzyme Immunoassay’ from Concept to Product Development. Duan, J. & Yuan, Z. 2001. Development of An Indirect Competitive Elisa for Ciprofloxacin

Residues in Food Animal Edible Tissues. Journal of Agricultural and Food Chemistry, 49, 1087‐1089.

Dufresne, G., Fouquet, A., Forsyth, D. & Tittlemier, S. A. 2007. Multiresidue Determination of Quinolone and Fluoroquinolone Antibiotics in Fish and Shrimp by Liquid Chromatography/Tandem Mass Spectrometry. Journal of AOAC International, 90, 604‐612.

Espinosa‐Mansilla, A., De La Pena, A. M., Gomez, D. G. & Lopez, F. S. 2006. Determination of Fluoroquinolones in Urine and Serum by Using High Performance Liquid Chromatography and Multiemission Scan Fluorimetric Detection. Journal of Talanta 68, 1215‐1221.

Fierens, C., Hillaert, S. & Van Den Bossche, W. 2000. The Qualitative And Quantitative Determination of Quinolones of First And Second Generation by Capillary Electrophoresis. Journal of Pharmaceutical and Biomedical Analysis, 22, 763‐772.

Garden, S. W. & Sporns, P. 1994. Development and Evaluation of An Enzyme Immunoassay for Sulfamerazine In Milk. Journal of Agricultural and Food Chemistry, 42, 1379‐1391.

Gaugin, M. & Abjean, J. P. 1998. Screening of Quinolone Residues in Pig Muscle by Planar Chromatography. Journal of Chromatographia, 47 (1‐2), 101‐104.

135

Gigosos, P., Revesado, P. R., Cadahia, O., Fente, C. A., Vazquez, B. I., Franco, C. M. & Cepeda, A. 2000. Determination of Quinolones in Animal Tissues And Eggs By HPLC with Photodiode‐Array Detection. Journal of Chromatography, 871, 31‐36.

Giroud, F., Gorgy, K., Gondran, C., Cosnier, S., Pinacho, D. G., Marco, M. P. & Sanchez‐Baeza, F. J. 2009. Impedimetric Immunosensor Based on A Polypyrrole‐Antibiotic Model Film for The Label‐Free Picomolar Detection Of Ciprofloxacin. Analytical Chemistry, 81, 8405–8409.

Goodrow, M. H. & Hammock, B. D. 1998a. Hapten Design For Compound‐Selective Antibodies: ELISAs for Environmentally Deleterious Small Molecules. Analytica Chimica Acta, 376, 83‐91.

Goodrow, M. H., Sanbom, J. R., Stoutamire, D. W., Gee, S. J. & Hammock, B. D. 1995. Strategies For Immunoassay Hapten Design. in Immunoanalysis of Agrochemicals ; Nelson, J. O., Karu, A. E., Wong, R. B., Eds.; Acs Symposium Series 586; . American Chemical Society, Pp.119‐139.

Haasnoot, W., Gercek, H., Cazemier, G. & Nielen, M. W. F. 2007. Biosensor Immunoassay for Flumequine in Broiler Serum and Muscle. Analytica Chimica Acta, 586(1‐2), 312‐318.

Hammer, P. & Heeschen, W. 1995. Antibody‐Captured Immunoassay for The Detection of ENR in Raw Milk. . Milchwissenschaft 50, 513‐514.

Harlow, E. & Lane, D. 1988. Antibodies : A Laboratory Manual. Hassonuan, M. K., Ballesteros, O., Taofiki, J., Vilchez, J. L., Cabrera‐Aguilera, M. & Navalon,

A. 2007a. Multiresidue Determination of Quinolones Antibacterials in Eggs of Laying Hens By Liquid Chromatography with Fluorescence Detection. Journal of Chromatography, 859(2), 282‐288.

Hassonuan, M. K., Ballesteros, O., Zafra, A., Vilchez, J. L. & Navalon, A. 2007b. Multiresidue Method for Simultaneous Determination of Quinolones Antibacterials in Pig Kidney Samples By Liquid Chromatography With Fluorescence Detection. Journal of Chromatography, 859(2), 282‐288.

Henniona, M. C. & Barcelo, D. 1998. Strengths and Limitations of Immunoassays For Effective and Efficient Use for Pesticide Analysis in Water Samples: A Review. Analytica Chimica Acta, 362 (1), 3‐34.

Hermanson, G. T. 1996. Bioconjugate Techniques. Hernandez‐Arteseros, J. A., Barbosa, J., Compano, R. & Prat, M. D. 2002. Analysis of

Quinolone Residues in Edible Animal Products. Journal Of Chromatography, 945, 1‐24.

Higgins, P. G., Fluit, A. C. & Schmitz, F. J. 2003. Fluoroquinolones: Structure And Target Sites. Current Drug Targets, 4, 181‐190.

Holtzapple, C. K., Buckley, S. A. & Stanker, L. H. 1997. Production and Characterization of Monoclonal Antibodies Against Sarafloxacin and Cross‐Reactivity Studies of Related Fluoroquinolones. Journal of Agricultural and Food Chemistry, 45, 1984‐1990.

Holtzapple, C. K., Buckley, S. A. & Stanker, L. H. 1999. Determination of Four Fluoroquinolones in Milk By On‐Line Immunoaffinity Capture Coupled with Reversed‐Phase Liquid Chromatography. Journal of AOAC International, 82, 607‐613.

Holtzapple, C. K., Buckley, S. A. & Stanker, L. H. 2001. Determination of Fluoroquinolones in Serum Using an On‐Line Clean‐Up Column Coupled to High‐Performance

136

Immunoaffinity–Reversed‐Phase Liquid Chromatography. Journal of Chromatography, 754, 1‐9.

Holtzapple, C. K., Pishko, E. J. & Stanker, L. H. 2000. Separation and Quantification of Two Fluoroquinolones in Serum by On‐Line High‐Performance Immunoaffinity Chromatography. Journal of Analytical Chemistry, 72, 4148‐4153.

Holtzapple, C. K. & Stanker, L. H. 1998. Affinity Selection of Compounds in a Fluoroquinolone Chemical Library By On‐Line Immunoaffinity Deletion Coupled to Column HPLC. Analytical Chemistry, 70, 4817‐4821.

Hooper, D. C. 2001. Mechanism of Action Of Antimicrobials: Focus on Fluoroquinolones. The Infectious Disease Society of America, 32, 9‐13.

Hooper, D. C. & Wolfson, J. S. 1991. Fluoroquinolone Antimicrobial Agents, The New England Journal of Medicine Editorial, 324, 384‐394.

Huang, B., Yin, Y., Lu, L., Ding, H., Wang, L., Yu, T., Zhu, J. J., Zheng, X. D. & Zhang, Y. Z. 2010. Preparation of High‐Affinity Rabbit Monoclonal Antibodies for Ciprofloxacin and Development of An Indirect Competitive Elisa for Residues in Milk. Journal of Zhejiang University‐Science B (Biomedicine & Biotechnology), 11(10), 812‐818.

Huet, A. C., Charlier, C., Singh, G., Godefroy, S. B., Leivo, J., Vehniäinen, M., Nielen, M. W. F., Weigel, S. & Delahauta, P. 2008. Development of an Optical Surface Plasmon Resonance Biosensor Assay For (Fluoro)Quinolones In Egg, Fish, And Poultry Meat. Analytica Chimica Acta, 623, 195–203.

Huet, A. C., Charlier, C., Tittlemier, S., Singh, G., Benrejeb, S. & Delahaut, P. 2006. Simultaneous Determination of (Fluoro)Quinolone Antibiotics In Kidney, Marine Products, Eggs, and Muscle by Enzyme‐Linked Immunosorbent Assay (Elisa). Journal of Agricultural and Food Chemistry, 54, 2822‐2827

Hung, S. 2007. New Detection Technique for Fluoroquinolone‐Conjugated Proteins by High Performance Liquid Chromatography with UV/Fluorescence Detectors. Journal of Food and Drug Analysis, 15, 71‐74.

Idowu, O. R. & Peggins, J. O. 2004. Simple, Rapid Determination of ENR and Ciprofloxacin in Bovine Milk and Plasma by High‐Performance Liquid Chromatography with Fluorescence Detection. Journal of Pharmaceutical and Biomedical Analysis, 35, 143‐153.

Ikegawa, S. 1998. Separatory Determination of Diastereomeric Ibuprofen Glucuronides in Huma Urine by Liquid Chromatography/Electrospray Ionization‐Mass Spectrometry. Journal of Biomedical Chromatography, 12, 317‐321.

Johnston, L., Mackay, L. & Croft M. 2002. Determination of Quinolones and Fluoroquinolones in Fish Tissue and Seafood by High Performance Liquid Chromatography with Electrospray Ionisation Tandem Mass Spectrometric Detection. Journal of Chromatography, 982, 97‐109.

Juhel‐Gaugain, M. & Abjean, J. P. 1998. Screening of Quinolone Residues in Pig Muscle by Planar Chromatography. Chromatographia, 47, 101‐104.

Kato, M., Ihara, Y., Kasai, D. & Kodaira, T. 2008. The Development of Release‐Competitive Assay To Detect Residual Quinolones in Contaminated Milk’, Journal af Food and Agricultural Immunology, 19, 241‐246.

Kato, M., Ihara, Y., Nakata, E., Miyazawa, M., Sasaki, M., Tsukasa, K. & Nakazawa, H. 2007. Development of ENR Elisa Using A Monoclonal Antibody Tolerating An Organic

137

Solvent with Broad Cross‐Reactivity To Other Newquinolones. Journal of Food and Agricultural Immunology, 18, 179‐197.

King, D. E., Malone, R. & Lilley, S. H. 2000. New Classification and Update on the Quinolone Antibiotics. American Family Physician, 61, 2741‐2748.

Klotins, K. 2005. Antibiotic Use for Growth Improvement ‐ Controversy and Resolution. http://www.omafra.gov.on.ca/english/livestock/animalcare/amr/facts/05‐041.htm. Downloaded on May 12th 2008.

Kuijper, E. J., Van Dissel, J. T. & Wilcox, M. H. 2007. Clostridium Difficile: Changing Epidemiology And New Treatment Options. Current Opinion in Infectious Diseases, 20, 376‐383.

Lee, N. A. & Kennedy, I. R. 2007. Immunoassay, Food Toxicants Analysis: Techniques, Strategies and Development. Elsevier, Valencia, Spain.

Li, Y., Ji, B., Chen, W., Liu, C., Peng, C. & Wang, L. 2008. Production of New Class‐Specific Polyclonal Antibody for Determination of Fluoroquinolone Antibiotics by Indirect Competitive Elisa. Journal of Food and Agricultural Immunology, 19, 251‐264.

Lu, S. L., Zhang, Y., Liu, J., Zhao, C., Liu, W. & Xi, R. 2006. Preparation of Anti‐Pefloxacin Antibody and Development of An Indirect Competitive Enzyme‐Linked Immunosorbent Assay for Detection of Pefloxacin Residue in Chicken Liver. Journal of Agricultural and Food Chemistry, 54, 6995‐7000

Mandell, L. & Tillotson, G. 2002. Safety of Fluoroquinolones: An Update. Canadian Journal of Infectious Diseases, 13, 54‐61.

Maraschiello, C., Cusidó, E., Abellán, M. & Vilageliu, J. 2001. Validation of An Analytical Procedure for the Determination of the Fluoroquinolone Ofloxacin in Chicken Tissues. Journal of Chromatography B: Biomedical Sciences And Applications, 754, 311‐318.

Marazuela, M. D. & Moreno‐Bondi, M. C. 2004. Multiresidue Determination of Fluoroquinolones in Milk by Column Liquid Chromatography with Fluorescence and Ultraviolet Absorbance Detection. Journal of Chromatography A, 1034, 25‐32.

Marchesini, G. R., Haasnoot, W., Delahaut, P., Gercek, H. & Nielen, M. W. F. 2007. Dual Biosensor Immunoassay‐Directed Identification of Fluoroquinolones in Chicken Muscle by Liquid Chromatography Electrospray Time‐of‐Flight Mass Spectrometry. Analytica Chimica Acta, 259–268

Mehlhorn, A. J. & Brown, D. A. 2007. Safety Concerns with Fluoroquinolones. Annals of Pharmacotherapy, 41, 1859‐1866.

Mella M, S., Acuña L, G., Muñoz Q, M., Perez C, C., Labarca L, J., Gonzalez R, G., Bello T, H., Dominguez Y, M. & Zemelman Z, R. 2000. Quinolones: General Characteristics of Structure and Classification. Quinolonas: Aspectos Generales Sobre Su Estructura Y Clasificación, 17, 53‐66.

Mitchell, M. A. 2006. ENR. Journal of Exotic Pet Medicine, 15(1), 66‐69. Mitra, S. 2003. Sample Preparation Techniques In Analytical Chemistry. Mock, W. L. & Ochwat, K. J. 2002. Substituent Effects in The Addition of Carboxylic Acids to

Arylcarbodiimides. Journal of Chemical Society, Perkin Transaction 2, 843‐847. Nelson, J. M., Chiller, T. M., Powers, J. H. & Angulo, F. J. 2007. Fluoroquinolone‐Resistant

Campylobacter Species and the Withdrawal of Fluoroquinolones from Use in Poultry: A Public Health Success Story. Clinical Infectious Diseases, 44, 977‐980.

Neu, H. C. 1992. Quinolone Antimicrobial Agents Annual Review Medicine, 43, 465‐486.

http://www.omafra.gov.on.ca/english/livestock/animalcare/amr/facts/05%E2%80%90041.htm

138

Nikolayenko, I. V., Galkin, O. Y., Grabchenko, N. I. & Spivak, M. Y. 2005. Preparation of Highly Purified Human Igg, Igm, and Iga for Immunization and Immunoanalysis. Ukrainica Bioorganica Acta 2, 3‐11.

Oliphant, C. M. & Green, G. M. 2002. Quinolones: A Comprehensive Review. American Family Physician, 65, 455‐464.

Pena, A., Silva, L. J. G., Pereira, A., Meisel, L. & Lino, C. M. 2010. Determination of Fluoroquinolone Residues In Poultry Muscle In Portugal. Analytical and Bioanalytical Chemistry, 397, 2615‐2621.

Price, C. P. & Newman, D. J. 1991. Principle And Practice of Immunoassay. Ridgway, K., Lalljie, S. P. D. & Smith, R. M. 2007. Sample Preparation Techniques for the

Determination of Trace Residues and Contaminants in Foods. Journal of Chromatography A, 1153, 36‐53.

Sárközy, G. 2001. Quinolones: A Class of Antimicrobial Agents. Veterinarni Medicina, 46, 257‐274.

Sheng, W., Xia, X., Wei, K., Li, J., Li, Q. X. & Xu, T. 2009a. Determination of Marbofloxacin Residues in Beef and Pork with An Enzyme‐Linked Immunosorbent Assay. Journal of Agricultural and Food Chemistry, 57, 5971‐5975.

Sheng, W., Xua, T., Maa, H., Wanga, X., Li, Q. & Li, J. 2009b. Development of An Indirect Competitive Enzyme‐Linked Immunosorbent Assay for Detection of Danofloxacin Residues in Beef, Chicken and Pork Meats. Journal of Food and Agricultural Immunology, 20, 35‐47.

Shim, J. H., Shen, J. Y., Kim, M. R., Lee, C. J. & Kim, I. S. 2003. Determination ff The Fluoroquinolone ENR in Edible Chicken Muscle by Supercritical Fluid Extraction And Liquid Chromatography With Fluorescence Detection. Journal of Agricultural and Food Chemistry, 7528‐7532.

Simonovska, B., Andrensek, S., Vovk, I. & Prosek, M. 1999. High‐Performance Thin‐Layer Chromatography Method for Monitoring Norfloxacin Residues on Pharmaceutical Equipment Surfaces. Journal of Chromatography A, 862, 209‐215.

Skerritt, J. H., Kurtz, D. A. & Stanker, L. 1995. New Frontiers in Agrochemical Immunoassay. AOAC International.

Smith, K. E., Besser, J. M., Hedberg, C. W., Leano, F. T., Bender, J. B., Wicklund, J. H., Johnson, B. P., Moore, K. A. & Osterholm, M. T. 1999. Quinolone‐Resistant Campylobacter Jejuni Infections in Minnesota, 1992–1998. The New England Journal of Medicine, 340(20), 1525‐1532.

Snitkoff, G. G., Grabe, D. W., Holt, R. & Bailie, G. R. 1998. Development of An Immunoassay For Monitoring Of The Level Ciprofloxacin In Patients Sample. Journal of Immunoasssy, 19(4), 227‐238.

Sun, W. Y., Liu, W. Y. & Qu, L. B. 2007. Development Of ELISA and Immunochromatographic Assay for Ofloxacin. Chinese Chemical Letters, 18, 1107‐1110.

Tittlemier, S. A., Gelinas, J. M., Dufrense, G., Haria, M., Queery, J., Cleroux, C., Menard, C., Delahaut, P., Singh, G., Durand, N. & Godefroy, S. 2008. Development of A Direct Competitive Enzyme‐Linked Immunosorbent Assay for The Detection Of Fluoroquinolone Residues In Shrimp. Journal Analytical Methods, 1, 28‐35.

Toldra, F. & Reig, M. 2006. Methods for Rapid Chemical and Veterinary Drug Residues in Animal Foods. Trends in Food Science and Technology, 17, 482‐489

139

Tollefson, L. & Karp, B. E. 2004. Human Health Impact from Antimicrobial Use in Food Animals. Médecine E T Maladies Infectieuses 34, 514‐521.

Toussaint, B., Chedin, M., Bordin, G. & Rodriquez, A. R. 2005a. Determination (Fluoro)Quinolone Antibiotic Residues in Pig Kidney Using Liquid Chromatography‐Tandem Mass Spectrometry: I. Laboratory‐Validated Method. Journal of Chromatography, 1088(1‐2), 32‐39.

Toussaint, B., Chedin, M., Vincent, U. & Rodriquez, A. R. 2005b. Determination (Fluoro)Quinolone Antibiotic Residues in Pig Kidney Using Liquid Chromatography‐Tandem Mass Spectrometry: Part Ii. Intercomparison Exercise. Journal of Chromatography, 1088(1‐2), 40‐48.

Tsekenis G., Garifallou, G. Z., Davis, F., Millner, P. A., Pinacho, D. G., Baeza, F. S., Marco, M. P., Gibson, T. D. & Higson, P. J. 2008. Detection of Fluoroquinolone Antibiotics in Milk Via A Labeless Immunoassay Based Upon an Alternating Current Impedance Protocol. Journal of Analytical Chemistry 80, 9233‐9239.

Van Emon, J. M. 2010. Bioanalytical Methods for Food Contaminant Analysis. Journal of AOAC International, 93 (6), 1681‐1691.

Van Emon, J. M., Chuang, J. C., Trejo, R. M. & Durnford, J. 2007. Immunoassay and Other Bioanalytical. 1‐43.

Van Vyncht, G., Janosi, A., Bordin, G., Toussaint, B., Maghuinrogister, G., De Pauw, E. & Rodriguez, A. R. 2002. Multiresidue Determination of (Fluoro)Quinolone Antibiotics in Swine Kidney Using Liquid Chromatography‐Tandem Mass Spectrometry. Journal of Chromatography, 952.

Vega, M., Rios, G., Saelzer, R. & Herlitz, E. 1995. Analysis of Quinolonic Antibiotics By HPTLC. Oxolinic Acid Residue Analysis in Fish Tissue. Journal of Planar Chromatography, 8, 378‐381

Volmer, D. A., Mansoori, B. & Locke, S. J. 1997. Study Of 4‐Quinolone Antibiotics in Biological Samples by Short‐Column Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry. Analytical Chemistry, 69, 4143‐4155.

Wagman, A. S. & Wentland, M. P. M. 2007. Quinolone Antibacterial Agents. Comprehensive Medicinal Chemistry, 7, 567‐596.

Wang, S., Allan, R. D., Skerritt, J. H. & Kennedy, I. V. 1998. Development of A Class‐Specific Competitive Elisa For The Benzoylphenylurea Insecticides. Journal of Agricultural and Food Chemistry, 46 (8), 3330–3338.

Wang, Z., Zhu, Y., Ding, S., He, F., Beier, R. C., Li, J., Jiang, H., Feng, C., Wan, Y., Zhang, S., Kai, Z., Yang, X. & Shen, J. 2007a. Development of A Monoclonal Antibody‐Based Broad‐Specificity ELISA for Fluoroquinolone Antibiotics in Foods And Molecular Modeling Studies Of Cross‐Reactive Compounds. Journal of Analytical Chemistry, 79, 4471‐4483.

Wang, Z., Zhu, Y., Ding, S. Y., He, F. Y., Beire, R. C., Li, J. C., Jiang, H., Feng, C., Wan, Y., Zhang, S., Kai, Z., Yang, X. & Shen, J. 2007b. Development of A Monoclonal Antibody‐Based Broad‐Specificity ELISA for Fluoroquinolone Antibiotics in Foods and Molecular Modeling Studies of Cross‐Reactive Compounds. Journal of Analytical Chemistry, 79, 4471‐4483.

140

Watanabe, H., Satake, A., Kido, Y. & Tsuji, A. 2002. Monoclonal‐Based Enzyme‐Linked Immunosorbent Assay and Immunochromatographic Assay for ENR in Biological Matrices. Analyst, 127, 98‐103.

Watanabe H., Satake., Kido, Y. & Tsuji, A. 2002. Monoclonal‐Based Enzyme‐Linked Immunosorbent Assay and Immunochromatographic Assay for ENR in Biological Matrices. The Analyst, 127, 98‐103.

Wild, D. 1994. The Immunoassay Handbook. Yoon, Y., Westerhoff, P., Snyder, S. A. & Esparza, M. 2003. HPLC‐Fluorescence Detection

and Adsorption of Bisphenol A, 17β‐Estradiol, and 17α‐Ethynyl Estradiol on Powdered Activated Carbon. Water Research, 37, 3530‐3537.

Yorke, J. C. & Froc, P. 2000. Quantitation of Nine Quinolones in Chicken Tissues By High‐Performance Liquid Chromatography with fluorescence Detection, Journal of Chromatography, 882, 63‐77.

Yu, W., Yu‐Dong, S., Zhen‐Lin, X., Hong‐Tao, L., Hong, W. & Yuan‐Ming, S. 2010. Production and Identification of Monoclonal Antibody Against Flumequine and Development of Indirect Competitive Enzyme‐Linked Immunosorbent Assay. Chinese Journal of Analytical Chemistry, 38(3), 313‐317.

Zeng, Z., Dong, A., Yang, G., Chen, Z. & Huang, X. 2005. Simultaneous Determination of Nine Fluoroquinolones in Egg White and Egg Yolk by Liquid Chromatography with Fluorescence Detection. Journal of Chromatography B: Analytical Technologies in The Biomedical and Life Sciences, 821, 202‐209.

Zhao, C., Liu, W., Ling, H., Lu, S., Zhang, Y., Liu, J. & Xi, R. 2007. Preparation of Anti‐Gatifloxacin Antibody and Development An Indirect Competitive Enzyme‐Linked Immunosorbent Assay for The Detection of Gatifloxacin Residue in Milk. Journal of Agricultural and Food Chemistry, 55, 6879‐6884.

Zhao, S., Li, X., Ra, Y., Li, C., Jiang, H., Li, J., Qu, Z., Zhang, S., He, F., Wan, Y., Feng, C., Zheng, Z. & Shen, J. 2009. Developing and Optimizing An Immunoaffinity Cleanup Technique for Determination of Quinolones from Chicken Muscle. Journal of Agricultural and Food Chemistry, 57, 365‐371.

Zheng, Z., Dong, A. G., Yang, G. X., Chen, Z. L. & Huang, X. H. 2005. Simultaneous Determination of Nine Fluoroquinolones in Egg White And Egg Yolk by Liquid Chromatography with Fluorescence Detection. Journal of Chromatography, 821, 202‐209.

Zhu, Y., Li, L., Wang, Z., Chen, Y., Zhao, Z., Zhu, L., Wu, X., Wan, Y., He, F. & Shen, J. 2008. Development of An Immunochromatography Strip for the Rapid Detection Of 12 Fluoroquinolones in Chicken Muscle and Liver. Journal of Agricultural and Food Chemistry, 55, 5469‐5474.

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