Altschuh_Esonn06_p2

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BICD 140 Immunology Winter 2000Rod Langman email: [email protected]://www-biology.ucsd.edu/classes/bicd140.WI00/lecture00v01.htm

Organization of immunoglobulin genes

Gene rearrangements, transcription and sysnthesis for the heavy chain

FromKuby Immunology 4eby Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne.

W. H. Freeman & Co. and Sumanas, Inc. Immunologyhttp://www.whfreeman.com/immunology/

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Gene rearrangements, transcription and sysnthesis for a κ light chain

FromKuby Immunology 4eby Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne.

W. H. Freeman & Co. and Sumanas, Inc. Immunologyhttp://www.whfreeman.com/immunology/

CDR1(H1)

CDR2(H2)

CDR3(H3)

VH

V DJ C

CH1 CH2 CH3

CDR1(L1)

CDR2(L2)

CDR3(L3)

V J CVL CL

Relation between gene segments and protein domains

From: Cellular and Molecular Immunology Abul K. Abbas    Andrew H. Lichtman    Jordan S. Pober ; W.B. Saunders Company

Schematic drawing of an IgG

VL

VHCLVL

VH CL

CH2

CH1 CH1

CH2

CH3CH3

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Germline IgHEAVY CHAIN GENES

REARRANGEMENT ON ONE ALLELE

PRODUCTIVE NONPRODUCTIVE

µmRNA

µ HEAVY CHAIN

1. Inhibits rearrangement on other allele(allelic exclusion)

2. Stimulates κ gene rearrangement

REARRANGEMENT ONSECOND ALLELE

PRODUCTIVE NONPRODUCTIVE

µmRNA

µ HEAVY CHAIN

Stimulates κ gene rearrangement

CELL DEATH

Order of rearrangement and expression of Ig heavy chain (µ) genes

FROM:Cellular and Molecular Immunology Abul K. Abbas Andrew H. Lichtman Jordan S. Pober W.B. Saunders Company

H

250-1000412

10.000 - 40.000

1 - 4 x 107

5 - 10 x 104

109 - 1011

κ

25040

1000

λ

230

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Germline genes• V gene segments• J segments• D segments

Combinatorial joiningV x J (x D)

H-L chain associations• H x k• H x l

Total potential repertoire with junctional diversity

Mechanisms contributing to the generation of the primary antibody repertoire (mouse)

FromCellular and Molecular Immunology Abul K. Abbas    Andrew H. Lichtman    Jordan S. Pober    W.B. Saunders Company

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Somatic mutations in V genesBerek C, Milstein C. Mutation drift and repertoire shift in the maturation of the immune response. ImmunolRev. 1987 Apr;96:23-41. Review.

FromKuby Immunology 4eby Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne. W. H. Freeman & Co. and Sumanas, Inc. Immunologyhttp://www.whfreeman.com/immunology/

Genetic bases of antibody diversity

The Ab repertoire consists of all Abs that an individual can produce in response to immunizationwith different Ags. It corresponds to the number of B cell clones that exist in the individual. Theprimary repertoire (antibody diversity) is estimated to be > 109 clones.

The organization of immunoglobulin genes, their transcription and translation, illustrated for aheavy chain, explain the generation of antibody diversity: how a huge antibody repertoire isgenerated from a genome of limited size.

The rearrangements that lead to the primary repertoire occur during B cell maturation in the bonemarrow, and are antigen-independent. After B cell activation in the presence of Ag, somaticmutations in the V gene segments are responsible for the affinity maturation of Abs.

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Definitions

Antigen: a natural or synthetic substance recognized by the immune system, able to induce aimmune respond and/or able to react with a lymphocyte receptor or antibody.

Hapten: substance capable of reacting with antibodies but unable to induce their production,due to its small molecular weight. Haptens must be coupled to carrier proteins to induce aresponse.

Immunogen: substance able to induce a immune response

Epitope (=antigenic determinant = antigenic site): part of the antigen that interacts with theantibody combining site or paratope

Cross-reaction: interaction of an Ab with an Ag different from the one that induced itsproduction

Heterospecificity: ability of an Ab to bind tighter to an Ag different from the one thatinduced its production

Continuous and discontinuous protein epitopes:

Continuous epitopes are formed of residues that arecontiguous in sequence. They can be mimicked by ashort peptide.

Discontinuous epitopes are formed of residuesadjacent in the tri-D structure of the protein, butdistant in sequence.

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Epitopes ContinuousDiscontinuous

Protein antigen

Epitope 3

Epitope 4Epitope 5

Epitope 1

Epitope 2

Antibody 1

Antibody 2Antibody 3

Antibody 4

Antibody 5

Cross-reactivity

Protein antigenMutant protein antigen

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1FDLFischmann TO, Bentley GA, Bhat TN, Boulot G, Mariuzza RA, Phillips SE, Tello D, Poljak RJ.Crystallographic refinement of the three-dimensional structure of the FabD1.3-lysozyme complex at 2.5-A resolution.J Biol Chem. 1991 266:12915-20.

3HFMPadlan EA, Silverton EW, Sheriff S, Cohen GH, Smith-Gill SJ, Davies DR.Structure of an antibody-antigen complex: crystal structure of the HyHEL-10 Fab-lysozyme complex.Proc Natl Acad Sci U S A. 1989 86:5938-42.

3HFLDavies DR, Padlan EA, Sheriff S.Antibody-antigen complexes.Annu Rev Biochem. 1990 . 59:439-73. Review.

Three X-ray structures of lysozyme-antibody complexes (Lysozyme has a similar orientation in the three pictures)

3HFM 3HFL1FDL

Antibody production for laboratory use

• Polyclonal antibodies

• Monoclonal antibodies

• Recombinant antibody fragments

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How are Abs produced for laboratory use ?

Polyclonal antibodies. Abs are present in the serum of an immunized animal orhuman. Abs specific for a given Ag can be obtained by affinity purification.They are polyclonal, i.e. directed to different epitopes of the Ag, henceheterogeneous in sequence and binding affinity.

Monoclonal antibodies (mAb). The hybridoma technology allows theproduction of Abs specific for a single epitope of the Ag. Mabs arehomogeneous: same sequence and Ag binding affinity.

Recombinant antibody fragments. Molecular biology techniques allow theproduction of homogeneous Ab fragments, mostly scFv (single chain Fragmentvariable) and Fab (Fragment antigen binding) from an heterologous source(bacteria such as E. coli), greatly facilitating their production and manipulationfor biotechnological, medical or research applications.

Hybridoma technology

1. Immunisation of a mouse with the antigen

2. Fusion of B-lymphocytes (antibody forming cell isolated from the spleen) with tumor cells

3. Screen cells for antibody production

B-lymphocyte Tumor cell

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Recombinant antibody technology

Antibody producing clone Isolate the mRNA andprepare the cRNA

Insert into a vector

Transform bacteriaAntibody producing bacteria

Recombinant antibody fragments

VL

CL

VH

CH1

VL

CL

VH

CH1

CH3

CH2

CH3

CH2

VL

CL

VH

CH1

VL VH

IgG Fab Fv scFv scFv

VL VH VL VH

VL VH

Toxin,enzyme,tag…

Hayden MS, Gilliland LK, Ledbetter JA. 1997Antibody engineering.Curr Opin Immunol. 9:201-12. Review.

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Antigen-antibody interactions

Definitions

Affinity: strength of the interaction, resulting from the sum of attractive and repulsive forcesbetween two molecular surfaces.

Specificity: ability to discriminate between related binding sites.

Valence: number of paratopes or epitopes per molecule. Example: IgGs are bivalent.

Avidity: apparent affinity resulting from a multivalent binding.

Binding parameters

Ab + Ag Ab-Agka

kd

ka (kon) : association rate constant (M-1.s-1)kd (koff) : dissociation rate constant (s-1)Ka; Kd : equilibrium affinity constants (M-1; M)

Reaction rate: d[Ab-Ag]/dt = ka [Ab][Ag] – kd [Ab-Ag]

At equilibrium: d[Ab-Ag]/dt = 0 ka [Ab][Ag] = kd [Ab-Ag]

[ Ab-Ag] ka 1

[Ab] [Ag] kd Kd

Ka = = =

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1NMBAg=neuraminidase (468 residues)

2CGRAg=2 N-(P-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid

3HFLAg=lysozyme (129 residues)

X-ray structures of antigen-antibody complexes (space filling models)

2CGRGuddat LW, Shan L, Anchin JM, Linthicum DS, Edmundson AB. Local and transmitted conformational changes on complexationof an anti-sweetener Fab. J Mol Biol. 1994 Feb 11;236(1):247-74.

3HFLDavies DR, Padlan EA, Sheriff S. Antibody-antigen complexes. Annu Rev Biochem. 1990 . 59:439-73. Review.

1 NMBMalby RL, Tulip WR, Harley VR, McKimm-Breschkin JL, Laver WG, Webster RG, Colman PM. The structure of a complexbetween the NC10 antibody and influenza virus neuraminidase and comparison with the overlapping binding site of the NC41antibody. Structure. 1994 Aug 15;2(8):733-46.

Indirect (sandwich)

Antigen Add antibody preparation Secondary labeled antibody Detection of bound target

Separation

Antigen Add antigen preparation Secondary labeled antibody Detection of bound target

Separation

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Immunoassay formats

Antibody Add labeled biomolecules Detection of bound target

Direct

Indirect (sandwich)

Inhibition

Antibody 1 Add biomolecules Secondary antibody and labelDetection of bound target

Antibody Add biomolecules Add labeled targetDetect a decrease in response in the presence ofunlabeled target

Separation

Separation

References for 1-7

Textbooks

Cellular and Molecular ImmunologyAbul K. Abbas Andrew H. Lichtman Jordan S. Pober W.B. Saunders Company

Kuby immunologyBarbara A. Osborne, Richard A. Goldsby, Thomas J. KindtW H Freeman

Roitt's Essential Immunology , Tenth EditionIvan Roitt, Royal Free & University College Medical SchoolPeter J Delves, University College London

Web sites

http://bioweb.wku.edu/courses/Biol328/default.htmlDr. Cheryl D. Davis, Western Kentucky University,USAAn introductory study of the mammalian immune system

http://www-immuno.path.cam.ac.uk/%7Eimmuno/part1.htmlMike Clark, Cambridge University, Cambridge UKImmunoglobulin Structure/Function

http://www.whfreeman.com/kuby/Richard A. Goldsby, Thomas J. Kindt, Barbara A. OsborneKUBY, IMMUNOLOGY

http://www.antibodyresource.com/educational.html The antibody resource pagehttp://www.biochem.unizh.ch/plueckthun/ The Plückthun Lab Homepagehttp://www.bioinf.org.uk/abs/ Antibodies - Structure and Sequencehttp://www.els.net/els/ Nature Encyclopedia of Life Sciences

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Biosensors and bioassays

Separation DetectionInteractionA) Bioassay

B) BiosensorInteraction = signal (detection)

Recognition unit

Target

Separation

* washing

Immunoassays: labels and separation steps

Anti-target antibodyon solid phase

Add solution to analyze Separation Add labeled secondary interactant Detection of bound target

Recognition unit Target Secondary interactant Label

* filtration

* Isotopes : RIA (radioImmunoAssay)* Fluorophores : Fluorescence immunoassays* Enzymes : ELISA (Enzyme Linked Immuno Sorbent Assay)

Labels (examples)

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Bioassays

Bioassays use receptors (molecules with affinity for a target) for the identification or quantificationof biological material (target) in a sample. Technically the receptor is reacted with the sample ofinterest, followed by detection of the receptor-target complex. This step requires the separationbetween free and bound molecules, and generally the addition of further reagents.

Immunoassays. Antibodies are widely used as receptors because of their antigen binding affinityand specificity, and because of their diversity, potentially allowing to monitor any target.

Separation. The complex can be separated from the free molecules by filtration, precipitation or bywashing when one of the molecules is coupled to a solid phase.

Detection. The complex is detected by means of a reagent that is labeled, for example with aradioisotope (RIA: Radio Immuno Assay), a fluorophore (Fluorescence Immunoassay), or anenzyme that generates a colored product when the substrate is added (ELISA: Enzyme LinkedImmuno Sorbent Assay).

Assay formats. In direct or indirect assays, the formation of a complex between the receptor and thetarget produces a signal that increases with target concentration. In inhibition assays, the presence ofthe target prevents formation of a complex between the recognition element and a labeled target,resulting in a decrease in signal.

Definition of a biosensor

A biosensor is an analytical device comprising two elementsin spatial proximity:

• A biological recognition element able to interactspecifically with a target

• A transducer able to convert the recognition event into ameasurable signal

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Biosensor classification IFrom Rodriguez-Mozaz S, Marco MP, Lopez de Alda MJ, Barcelo D. Biosensors for environmental monitoring of endocrine disruptors: a review article. Anal Bioanal Chem. 2004 Feb;378(3):588-98. Review.

Biosensor classification II

Label free, general approaches• Surface Plamon Resonance• Calorimetric• Acoustic- Cooper MA. Label-free screening of bio-molecular interactions. Anal Bioanal Chem. 2003 Nov;377(5):834-42. Review.

Label free, system specific approaches• Enzymatic (catalytic) sensorsD'Orazio P. Biosensors in clinical chemistry. Clin Chim Acta. 2003 Aug;334(1-2):41-69. Review.Luque de Castro MD, Herrera MC. Enzyme inhibition-based biosensors and biosensing systems: questionableanalytical devices. Biosens Bioelectron. 2003 Mar;18(2-3):279-94. Review.

Label based, general approaches• Environmentally sensitive fluorophores• Redox compounds, enzymesHellinga HW, Marvin JS. Protein engineering and the development of generic biosensors. TrendsBiotechnol. 1998 Apr;16(4):183-9. Review.Warsinke A, Benkert A, Scheller FW. Electrochemical immunoassays. Fresenius J Anal Chem. 2000 Mar-Apr;366(6-7):622-34. Review.

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Biosensors

The definition of biosensors applied here is that given by Scheller et al. (2001): “ the direct spatialcombination of biological recognition and transduction into an electrical signal”. The transducerconverts a change in property of a solution or surface upon complex formation into an electricalsignal. As a consequence of the direct physical combination between the recognition and transducerelements, the interaction with the target is detected as it takes place.

Biosensors therefore differ from conventional bioassays by the fact that auxiliary procedures(addition of reagents, separation steps) are not required.

Classification Biosensors are generally classified according to transduction modes and recognitionelements (D’Orazio, 2003; Rodriguez-Mozaz et al., 2004). They can also be classified according totheir applicability (need or not for a label, general or not). Examples for each group will be given,with special emphasis on a label-free general approach (Biacore).

D'Orazio P. Biosensors in clinical chemistry. Clin Chim Acta. 2003 334:41. Review.

Rodriguez-Mozaz S, Marco MP, Lopez de Alda MJ, Barcelo D. Biosensors for environmental monitoring ofendocrine disruptors: a review article. Anal Bioanal Chem. 2004. 378:588.

Scheller FW, Wollenberger U, Warsinke A, Lisdat F. Research and development in biosensors. Curr OpinBiotechnol. 2001.12:35. Review.

SPR biosensors

- Principle of the Biacore technology- The phases of a sensorgram- The sensor surface- Preliminary steps for quantitative measurements- Experimental conditions and data interpretation- Application examples

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BIACORE instruments (Biacore SA, Uppsala, Sweden) are optical biosensors based on thephenomenon of surface plasmon resonance (SPR).

1

prismglassgold

Sensor Surface

Flow

Intensity

Angle

1

2

prismglassgold

2

Sensor Surface

Flow

RU

Temps

1

2

Sensorgram

Principle of the BIACORE technology

Principle of the Biacore technology

Biacore® SPR technology: http://www.biacore.com/technology/core.lasso

BIACORE instruments(Biacore AB, Uppsala, Suède) are biosensors based on the opticalphenomenon of surface plasmon resonance (SPR).

1

prismglassgold

2

Sensor surface

Continuous flow

Biological Receptor :Immobilized ligand

Transducer: SPR

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10

RU

Time (s)

Analyte injection phase

Post-injection phase

Regeneration

The phases of a sensorgram

ka (M-1.sec-1) kd (sec-1) Ka (M-1)

106 10-3 109

0

50

100

150

200

250

0 120 240 360 480 600

Response(RU)

Time (seconds)

(PM 50000, Rmax 200 Rus, Flow 10 µl/mn, A= 50 nM)

104 10-5 109

105 10-4 109

107 10-2 109

Simulated kinetics for Ka = ka/kd = 109 M-1

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SPR biosensors: the Biacore technology(Biacore AB, Uppsala, Sweden; http://www.biacore.com).

Biacore instruments are biosensors that use the optical phenomenon of surface plasmon resonance

(SPR) to monitor molecular interactions in real time (Malmquist and Karlsson, 1997; Rich and

Myszka, 2003). One of the interactants (called the ligand) is immobilized on the dextran matrix of a

sensorchip. The second interactant (called the analyte) is injected in a continuous flow by means of

a microfluidic system. Molecular association and dissociation events produce variations in the SPR

signal that are recorded as resonance units (RU) as a function of time. The mathematical evaluation

of the resulting curves called sensorgrams, allows to calculate the kinetic parameters and the

equilibrium affinity of the ligand-analyte interaction.

Malmqvist M, Karlsson R. Biomolecular interaction analysis: affinity biosensor technologies for functional

analysis of proteins. Curr Opin Chem Biol. 1997. 1:378-83. Review

Rich RL, Myszka DG. A survey of the year 2002 commercial optical biosensor literature. J Mol Recognit.2003.16:351.

Glass

GoldLinker

Carboxylated dextran(non cross-linked)

Ligand

The sensor surface

Properties:* Aqueous environnement (hydrogel containing 97-98% water)* Mobility (chains are not cross-linked)* Efficient use of the evanescent field (thickness about 100 nm)* Increased sensitivity (more coupling sites than on a flat surface)* Allows covalent coupling (through carboxyl groups)

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Sensor surfaces. The standard Biacore sensor surface (CM5) is a glass slide with a goldlayer, covered with a carboxymethylated dextran matrix in which the interactions take place.

A number of other surfaces are available :http://www.biacore.com/technology/sensorchips.lasso

Preliminary steps for measuring surface interactions

1. Immobilization of one interactant

2. Regeneration conditions

3. Reference surface

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1. Immobilization of the ligand

* Captured ligand- streptavidin/biotin- NTA/His-tag- hydrophobic surfaces/lipids- anti-antibody/antibody- anti-GST/GST(fusion proteins)- anti-peptide/peptide (fusion proteins)

Analyte

LigandCapture

Analyte

Ligand

* Covalent- amine coupling- thiol coupling

Immobilization chemistry : amine coupling

CONHCH2CH2OH

COO-

COONHProt

COO-

COO-

COO-

COON

COO-

COON

O

OO

O

COON

COO-

COONHProt

O

O

EDC/NHS H2N-Prot Ethanolamine

EDC N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochlorideNHS N-hydroxysuccinimideEthanolamine-HCl H2NCH2CH2OH

Resp

onse

(RU

)

Time (sec)Time (s)400 800 1200

35000

30000

25000

20000

15000

1600

Res

pons

e (R

U)

EDC/NHS

Lig

and

Ethanolamine

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2. Regeneration

Objectives:

* Total analyte dissociation (baseline stability)

* Conserved ligand activity (reproducibility of analyte response)

Examples of regeneration solutions

Hydrochloric acid 10-100 mMPhosphorique acid 10-100 mMGlycine-Hcl 1.5M pH 2.5Sodium hydroxide 10-100 mMEthanol 25-70%Sodium chloride 1MAcetonitrile 5-10%...

Inject in ‘pulses’ = < 1 mn

Check compatibility with instrumentation (Instrument Handbook)

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13500

14000

14500

15000

15500

16000

16500

17000

17500

0 40 80 120 160 200 240 280 320 360 400Time s

Resp

on

se

RU

-200

0

200

400

600

800

1000

1200

1400

1600

0 50 100 150 200 250Time s

Resp

on

se

RU

Injection Post-injection Régénération

Injection Post-injection

3. Reference surface

Different factors affect the SPR signal:• Binding (spscific or aspecific) of molecules to the sensor surface• Buffer composition• Temperature• Pressure• Compactness of dextran matrix• …..

The ideal reference surface should mimic the physical properties of the ligandsurface, but not its binding properties

Which reference surface ?

Ligand surface-specific binding to the ligand-aspecific binding to the ligand-aspecific binding to the surface

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The reference surface should mimic the physico-chemical properties of the ligandsurface, but not its analyte binding properties

0

50

100

150

200

250

0 120 240 360 480 600

Response(RU)

Time (seconds)

Analyte injectionphase

0

50

100

150

200

250

0 120 240 360 480 600

Response(RU)

Time (seconds)

Ligand surface

Reference surface

After deducingthe reference

Evaluation of kinetic data: experimental and theoretical curves

-10

0

10

20

30

40

50

60

70

80

90

-10 30 70 110 150 190 230 270 310 350

Time s

Resp

on

se

RU

ka (1/Ms) kd (1/s) Rmax (RU) KA (1/M) KD (M) Chi29,41E+05 1,67E-03 65,6 5,65E+08 1,77E-09 0,124

25

0

400

800

0 200 400 600 800Time (s)

Res

po

nse

(R

U)

Injection Post- injection

Reference surface Ligand surface

Time (s)

Res

po

nse

(R

U)

0

400

800

1200

0 400 800 1200

Injection Post- injection

Legend:

E6

GST-Peptide

Antibody

-100

0

100

200

0 200 400 600 800Time (s)

Res

po

nse

(R

U)

Reference surface Ligand surface

A

B C

Preliminary steps for quantitative measurements

Immobilization of the ligand. The ligand can be immobilized covalently or captured on thesurface through a first covalently immobilized capture molecule (Abs directed to Abs or tag,streptavidine…).

Regeneration of the surfaces. A successful surface regeneration allows the same surface (with acovalently immobilized ligand or capture molecule) to be reused several times.

Reference surfaces. The quality of biosensor data can be greatly improved by using appropriatereference surfaces and buffer correction procedures (Karlsson and Stahlberg, 1995; Karlsson andFält, 1997; Myszka, 1999), which eliminate artifacts due to bulk refractive index changes,aspecific binding, injection noise, matrix effects, and baseline drift.

Karlsson R, Stahlberg R. Surface plasmon resonance detection and multispot sensing for direct monitoring ofinteractions involving low-molecular-weight analytes and for determination of low affinities. Anal Biochem.1995. 228:274.

Karlsson R, Falt A. Experimental design for kinetic analysis of protein-protein interactions with surfaceplasmon resonance biosensors. J Immunol Methods. 1997. 200:121.

Myszka DG.Improving biosensor analysis. J Mol Recognit. 1999. 12:279.