Bioassay Syed Shariq Naeem
May 26, 2015
Bioassay
Syed Shariq Naeem
Outline
• Types of assays• Introduction• Definition• Indication and principles of bioassay • Basic procedure• Calculations • Source of errors• Summary
Types of Assays
• Biological assays• Chemical assays: – Spectrophotometer, – Spectrofluorimetry, – Chromatography,
• Radio Immunoassays• Microbiological assays
Introduction
• Late 18th centaury- standardization of Diphtheria antitoxin by Paul Ehrlich
• Bioassay literal meaningo Bio – living tissueo Assay- assessment / measuremento Bioassay: Assessment of a biological substance
Definition
Comparative assessment of relative potency of a test compound to a standard compound on a living or biological tissue.
Quantitative measurement of the amount of active principle or substance in a pharmaceutical preparation or biological material using a suitable biological system
Indications for Bioassay• Active principle of drug is unknown• Active principle cannot be isolated, e.g. insulin, posterior pituitary
extract etc. • Chemical method is either – not available – if available, too complex, – insensitive to low doses e.g. Histamine can be assayed in microgram conc.
• Chemical composition of drug is different but has same pharmacological action e.g. cardiac glycosides isolated from diff sources, catecholamines etc.
• To measure LD 50 and ED 50• For biological standardization of drugs from natural sources which
cannot be obtained in a chemically pure form e.g., vasopressin, oxytocin, insulin, heparin
Principles of bioassay
• Bioassay involves the comparison of the main pharmacological response of the unknown preparation with that of the standard.
• The reference standard and test sample should have same pharmacological effect and mode of action, so that their DRC curve run parallel and their potency ratio can be calculated.
• The test solution and standard should be compared for their established pharmacological effect using a specified pharmacological technique.
• The method selected should be reliable, sensitive, reproducible and should minimize errors due to biological variation and methodology. ( Animals should of same species, sex and weight and number of animals should be large enough to permit statistical analysis.)
Procedure 1. Prepare the physiological salt solution2. Arrange the instrument and adjust the water bath.3. Balance the lever4. Tissue selection5. Surgical process and collection of required tissue.6. Tissue attachment to the water bath7. Relaxation time given to the tissue8. Prepare the standard drug( serial dilution)9. Select the lowest possible measurable concentration by trial
and error method.10. Prepare DRC for the standard drug.11. Prepare DRC for the test drug.( serial dilution)12. Select a assay method (3 point or 4 point assay)13. Calculation
Step 1: Prepare the physiological salt solution
Various Physiological salt solutionsFrog-Ringer
Kreb’s Tyrode Ringer-Locke
De Jalon
Mc Ewen
NaCl 65 g 69 g 80 g 91.5 g 90 g 76 g
KCl 1.4 g 3.5 g 2.0 g 4.2 g 4.2 g 4.2 g
MgCl². 6H²O --- 1.1 g 1.0 g --- --- ---
NaH2PO4. H²O 0.1 g 1.4 g 0.5 g --- --- 1.4 g
NaHCO³ 2 g 21 g 10 g 1.5 g 5 g 21 g
CaCl² 1.2 g 2.8 g 2 g 2.4 g 0.6 g 2.4 g
Glucose 20 g. 20 g. 10 g. 10 g. 5 g. 20 g
Aerating Gas air O² + 5%CO²
O² or air Pure O² O² + 5% CO²
O² + 5% CO²
For 10 litrespH- 7.3-7.4
•Calcium chloride to be added last.•Calcium chloride and magnesium chloride are hygroscopic, so use stock solution.
Uses: Physiological salt Solutions
Physiological salt solutions
Uses
Frog-Ringer Amphibian tissue preparation
Kreb’s Mammalian/Avian skeletal muscle preparation
Tyrode Intestine preparation
Ringer-Locke Heart muscle preparation
De Jalon Rat uterus preparation
Electrolytes Ingredients Functions
NaCl Maintain osmolarity
K+ Nerve conduction, muscle contraction, maintain heart rate & rhythm
Ca + Contraction
Mg+ Neurotransmission , decrease spontaneous activity
NaHCO³ & NaH2PO4 Buffer
Glucose Nutrient
Step 2: Arrange the instrument and adjust the water bath.
Kymograph: Sherrington- starling kymograph To obtain a graphical amplified
measurable response of a muscle or tissue
Two important parts: motor box and drum
Speed lever: 1 revolution/ 96 min. Paper:
glossy side outside – least resistance Rough side inside – stick to the
drum. Fixing solution: shellac and
colophony saturated in alcohol
Student Organ bath
• Outer bath:- First designed by rudolph
magnus Perpex glass Store water outside the
inner bath to maintain the temperature
• Inner bath:-– Glass– To observe the tissue during
experiment – 5-50ml (usually 10ml)
• Tissue holder and oxygen supply:- Tissue is attached inside the inner water bath to a
tissue holder. Also supports the oxygen supply to the tissue.
Step:3 -Balance the lever
• Lever: Three basic parts:• Effort arm- where force in
applied• Load arm- where effect of
force is observed• Fulcrum
Classes of lever – 3
Types of lever
• Magnification := Distance from the fulcrum to the writing point
Distance form the fulcrum to the tied tissue
o For slow contracting muscles:- 10-15 timeso For fast contracting muscles:-5-10 times
Step:4-Tissue selection
S.No Compound Tissue used
1. Acetylcholine Guinea-pig ileumFrog rectus abdominis muscleLeech dorsal muscleRat uterus preparationIsolated guinea-pig auricles
2. Serotonin Isolated oestrous uterus of ratIsolated fundic strip of ratGuinea pig ileum Rabbit ear preparationIsolated heart of the mollusc Venus mercenaria
S. No Chemical Tissue used
3. Histamine Guinea pig isolated ileum Guinea pig tracheal chain. Fall in BP of dog/cat
4. Adrenaline and noradrenalin
Rat colonNon pregnant rat uterusRat fundusRabbit aortic stripRabbit jejunumTracheal chain of guinea pig
Step 5: Surgical process and collection of required tissue.
• Animal sacrificed by cervical dislocation.• Tissue identified and isolated.• Carefully dissect and separate unwanted tissue.• Tissue kept in a physiological salt solution.• Avoid excessive handling of tissue.
Step 6 : Tissue attachment to the water bath
• Attach the ends of the tissue:-– One end:- tissue holder– Other end:- lever
• Method of attachment of tissue:– Attach the thread at the end by a needle – Intestine:- care should be taken not to block the lumen
Aeration
Pure oxygen (O2 ) For heart
Air For intestine
Carbogen ( 95% O2 & 5% CO2 )
For uterus
Mixing of the test drugHomogenisation of the solutionKeeping the tissue lumen patentTo maintain pH ( aeration by pure O2 causes losing of CO2 & solution becomes alkaline )
Temperature
Rabbit intestine Physiological temp.(37°C ) is needed for mammalian tissues
Guinea-pig ileum Temp. should be decreased in some experiment to decrease spontaneous contractions
Frog rectus muscle Amphibian tissue can survive in room temperature
Temperature should be constant through out the experiment
Step 7:Relaxation time given to the tissue
1. Intestine 30-45 min
2. Frog rectus 45-60 min
Measures to decrease spontaneous contraction:-Hanging a weight of appropriate amountGiving a antagonist
oE.g. Acetylcholine for blocking spontaneous contraction of ileum.
Step 8: Prepare the standard drug( serial dilution)
• Serial dilution: 10---10-9
Step 9: Prepare DRC for the standard and test drug
• Select two std doses s1& s2 from linear part of DRC [ Let the corresponding response be S1, S2]• Also s2/s1 = t2/t1 = 3/2
Time cycle
Contact timeTime allowed for the drug (agonist) to remain in contact with the tissue
Frog rectus abdominis muscle Guinea-pig ileum
90 sec 30 sec
Time ( min )
Event
0 Start the kymograph
2 Add the Acetylcholine
2.5 Stop the kymograph & wash the preparation
10 Wash the preparation
15 Start the kymograph
Start kymogarp
h
Add Ach
Stop kymograp
h
Wash preparatio
n
Wait for 11.5 min
Step 10: Perform a assay (3 or 4 point assay)
Types of Bioassays
• [1] Quantal Assays [ Direct endpoint ] Elicits an ‘All or None’ response in different
animals E.g.
Digitalis induced cardiac arrest in guinea pigs Hypoglycaemic convulsions in mice. Digitalis induced head drop in rabbits
• [2] Graded Response Assays Graded responses to varying doses Unknown dose response measured on same tissue
[2] Graded Response Assays [ Direct comparison on same tissues]
Interpolation: Conc. of unknown is
read from a standard plot of a log dose response curve of at least 4 sub maximal concentrations
Matching & Bracketing: Const dose bracketed with varying doses of standard till
exact match is obtained• Used when test sample is too small• Inaccurate & margin of error difficult to estimate• Eg histamine on guinea pig ileum, Posterior pituitary on rat uterus
Multiple Point Assays• 3 point assay
• 4 point assay
4 point assay [2 +2 dose assay]• Procedure [E.g. Ach bioassay]
Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test solution
Select two std doses s1& s2 from linear part of DRC [ Let the corresponding response be S1, S2]
Choose two test doses t1 & t2 with response T1 &T2 between S1 & S2 ; Also s2/s1 = t2/t1 = 2/3 Record 4 data sets [Latin square: Randomisation reduces error]
• s1 s2 t1 t2• s2 t1 t2 s1• t1 t2 s1 s2• t2 s1 s2 t1
3 point assay [2+1 dose assay]
• Fast & convenient• Procedure [E.g. Ach bioassay]
Log dose response [LDR] curve plotted with varying conc of std Ach solutions and given test solution
Select two std doses s1& s2 [ in 2:3 dose ratio] from linear part of LDR [ Let the corresponding response be S1, S2]
Choose a test dose t with a response T between S1 & S2 Record 4 sets data [Latin square: Randomisation reduces
error] as follows s1 s2 t t s1 s2 s2 t s1 s1 s2 t
Log Potency ratio [ M ] = [ (T –S1) / (S2-S1) ] X log d [d = dose ratio]
Step 11: Calculation
• Calculate the height of each response.• Take mean of all S1, S2, T1 and T2 values.
• Plot a graph
S1
S2
T1
T2
D1 D2
M
S1
S2
T1
T2
D1 D2
Calculation of the strength of the solution from graph :
• We know that D1=D2• EG..• 0.675 ml of 1 µg/ml= 0.425 of D2 conc.• D2 = 0.675/ 0.425 = 1.59 of 1 µg/ml• Strength of D2 = 1.59 µg/ml
Log potency ratio :• The horizontal separation M of the two curves
represents the log potency ratio of the concentration of test solution and of standard
Direct calculations• M={(T1-S1) +(T2 –S2)}/{(S2-S1) +(T2-T1)}×log d• Log d = log[s1/s2]
Where,• M = Potency of the drug• S1 & S2 = Length of the standard dose
response selected between 25-75 %• T1 & T2 = Length of the test drug response• s1 & s2 = Standard drug dose which came in
contact with tissue and had given the response S1 & S2 respectively
• Dilution of the inner water bath has to be taken in to account
• Strength of test solution = s1/t1 × antilog of M
• Dilution of the inner water bath has to be taken in to account
Calculation of the percentage error:-• Percentage error = ACT-OCT × 100
ACTWhere,• ACT = Actual concentration of test• OCT = Observed concentration of test
• The permissible limit of percentage error is <10%
Errors in bioassays
• Margin of error of bioassay should be < 10%
• Two types:-1. Biological variation: 2. Methodological variation
• Biological variation:-1. Variation in response to a drug. 2. Down regulation of receptor (repeated washing
of tissue)3. Loss of tissue sensitivity (change the tissue)4. Laboratory condition may be variable.
• Methodological variation:-1. Human error: done by the experimenter2. Experimental error: faulty procedure selection or
calibration error.(proper balancing the lever, and by maintaining the ph and temperature at a physiological level.)
• Reasons for methodological error:1. Lack of standardization of procedure2. Over handling of tissue 3. Preparation of physiological salt solution.4. Drug preparation or in dilution
Prepare Physiological sol.
Check instruments
Relaxation
Prepare DRC and Do a 4 point assay
Summary
Thank you
Summary 1. Prepare the physiological salt solution2. Arrange the instrument and adjust the water bath.3. Balance the lever4. Tissue selection5. Surgical process and collection of required tissue.6. Tissue attachment to the water bath7. Relaxation time given to the tissue8. Prepare the standard drug( serial dilution)9. After relaxation test any concentration of the drug10. Then standardize the tissue response with same drug. ( take subsequent two
response)11. Select the lowest possible measurable concentration by trial and error
method.12. Prepare DRC for the standard drug.13. Prepare DRC for the test drug.( serial dilution)14. Select a assay method (3 point or 4 point assay)15. Measure the height of each response16. Calculation
Time cycle
Time ( mins) Event
0 Raise the 1 gm weight & start the kymograph
2 Add acetylcholine
3.5 Stop the kymograph, wash rectus & lower the 1 gm weight
6 Raise the weight & start the kymograph
Contact timeTime allowed for the drug (agonist) to remain in contact with the tissue
Frog rectus abdominis muscle Guinea-pig ileum
90 sec 30 sec
Principles of Bioassay• Active principle to be assayed should show the same
measured response in all animal species• The degree of pharmacological response produced should
be reproducible under identical conditions [Eg Adrenaline shows same rise in BP in the same species under identical conditions: wt, age, sex, strain / breed etc]
• The reference standard must owe its activity to the principle for which the sample is being bioassayed
• Activity assayed should be the activity of interest• Individual variations must be minimised / accounted for• Bioassay might measure a diff aspect of the same substance
compared to chemical assay [Eg testosterone & metabolites
Biological objects
Whole animal
Isolated organ Isolated tissue
Isolated cells
Assay of insulin in rabbits
Assay of gonadotropins on ovary
Assay of oxytocin on isolated uterine tissue
Assay of antibiotics on bacterial cells