Determination of antibody affinity and kinetic binding constants in Gyrolab Bioaffy microfluidic CD

69 Pages·2008·1.53 MB·English

Department of Physics, Chemistry and Biology

Master Thesis

Determination of antibody affinity and kinetic
binding constants in Gyrolab Bioaffy microfluidic CD

Mikael Karlsson

Performed at Gyros AB

2008-04-01

LITH-IFM-A-EX-08/1931-SE

Linköping University, Department of Physics, Chemistry and Biology
581 83 Linköping, Sweden

Department of Physics, Chemistry and Biology

Master Thesis

Determination of antibody affinity and kinetic
binding constants in Gyrolab Bioaffy microfluidic CD

Mikael Karlsson

Performed at Gyros AB

2008-04-01

Supervisors
Johan Engström
Stephan Hoffmann
Gyros AB

Examiner
Olle Inganäs
Department of Physics, Chemistry and Biology
Linköping University

Datum
Avdelning, institution
Division, Department Date

Biomolecular and Organic Electronics 2008-04-01
Department of Physics, Chemistry and Biology
Linköping University

Språk Rapporttyp ISBN
Language Report category

Svenska/Swedish Licentiatavhandling ISRN: LITH-IFM-A-EX-08/1931-SE
Engelska/English Examensarbete
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URL för elektronisk version
http://urn.kb.se/resolve?urn=urn:nbn:s
e:liu:diva-11616
Titel
Title

Determination of antibody affinity and kinetic binding constants in Gyrolab Bioaffy
microfluidic CD

Författare
Author

Mikael Karlsson

Sammanfattning
Abstract

Studies of binding reactions are of highest importance in a vast number of areas of biomedicine
and biotechnology. A demand for fast and accurate small-volume measurements grows stronger,
partly due to the development of therapeutic antibodies. In this report, a novel method for studies
of binding reactions of antibodies is described. The use of a microfluidic platform shows
promising results in determination of affinity binding constants.

Affinities between 1*10-09 and 1*10-11 M have been determined for four TSH antibodies.
Reproducibility tests give a CV below 10%, using different Gyrolab instruments and microfluidic
CD:s. The method carries the advantages of using solution-based measurements of unmodified
molecules. Also an initial proof-of-concept for measurement of binding reaction rate constants
shows further usage of the method. The kinetic association rate constant has been determined to
2*106 M-1s-1 for one antibody. The possibility of using this method for screening of antibody
libraries is also discussed.

Nyckelord
Keyword

Affinity constants; Equilibrium dissociation constants, Kinetic rate constants; Immunoassay, Monoclonal antibodies, Microfluidics;
Gyrolab Bioaffy; Microlaboratory; Compact disc

Abstract
Studies of binding reactions are of highest importance in a vast number of areas of
biomedicine and biotechnology. A demand for fast and accurate small-volume
measurements grows stronger, partly due to the development of therapeutic
antibodies. In this report, a novel method for studies of binding reactions of antibodies
is described. The use of a microfluidic platform shows promising results in
determination of affinity binding constants.

Affinities between 1*10-09 and 1*10-11 M have been determined for four TSH
antibodies. Reproducibility tests give a CV below 10%, using different Gyrolab
instruments and microfluidic CD:s. The method carries the advantages of using
solution-based measurements of unmodified molecules. Also an initial proof-of-
concept for measurement of binding reaction rate constants shows further usage of the
method. The kinetic association rate constant has been determined to 2*106 M-1s-1 for
one antibody. The possibility of using this method for screening of antibody libraries
is also discussed.

Table of contents
COMMON ABBREVIATIONS..................................................................................................................1
1 INTRODUCTION.................................................................................................................................3
1.1 AIM...................................................................................................................................................3
2 THEORY................................................................................................................................................5
2.1 ANTIBODIES......................................................................................................................................5
2.1.1 Antibody Structure and Therapeutic Use................................................................................5
2.1.2 Antibody Affinity.....................................................................................................................6
2.1.3 Effects Influencing on Affinity Measurements........................................................................7
2.1.4 Studying Affinity.....................................................................................................................7
2.2 IMMUNOASSAYS................................................................................................................................8
2.3 MICROFLUIDIC ANALYSIS SYSTEMS.................................................................................................9
2.4 GYROS’ ANALYSIS PLATFORM..........................................................................................................9
3 ANALYSIS METHOD........................................................................................................................13
3.1 THE IMMUNOASSAY........................................................................................................................13
3.2 REAGENT SYSTEM...........................................................................................................................15
3.3 ANALYSIS EVALUATION.................................................................................................................16
3.4 AFFINITY DETERMINATION.............................................................................................................16
3.4.1 Mathematical Model.............................................................................................................16
3.4.2 Alternative Mathematical Model..........................................................................................18
3.4.3 Reaching Equilibrium...........................................................................................................19
3.5 KINETIC MODEL..............................................................................................................................19
3.6 RESULT INTERPRETATION AND REGRESSION METHOD...................................................................21
4 EXPERIMENTAL SECTION...........................................................................................................25
4.1 MATERIALS AND ASSAY REAGENTS...............................................................................................25
4.2 PREPARATION OF DETECTING AND CAPTURE REAGENTS................................................................25
4.3 STANDARD CURVE..........................................................................................................................26
4.4 UNSPECIFIC INTERACTIONS.............................................................................................................26
4.5 MODELLING WITH MATLAB...........................................................................................................26
4.6 EQUILIBRIUM TIME ESTIMATION....................................................................................................27
4.7 FIRST AFFINITY MEASUREMENTS...................................................................................................27
4.8 CAPTURE REAGENT STUDY.............................................................................................................27
4.9 AFFINITY MEASUREMENTS.............................................................................................................28
4.10 REPEATABILITY TEST.....................................................................................................................28
4.11 CONCENTRATION INFLUENCE ON KD..............................................................................................28
4.12 KINETIC CONSTANTS......................................................................................................................28
5 RESULTS AND DISCUSSIONS........................................................................................................29
5.1 PREPARATION AND EVALUATION OF REAGENTS.............................................................................29
5.2 STANDARD CURVE..........................................................................................................................29
5.3 UNSPECIFIC BINDING......................................................................................................................32
5.4 MODELLING WITH MATLAB............................................................................................................33
5.5 EQUILIBRIUM TIME ESTIMATION....................................................................................................34
5.6 FIRST AFFINITY MEASUREMENTS...................................................................................................36
5.7 CAPTURE REAGENT STUDY.............................................................................................................37
5.8 ADDITIONAL AFFINITY MEASUREMENTS........................................................................................38
5.9 REPEATABILITY TEST.....................................................................................................................39
5.10 CONCENTRATION INFLUENCE ON KD..............................................................................................40
5.11 KINETIC CONSTANTS......................................................................................................................42
6 FURTHER DISCUSSIONS................................................................................................................45
6.1.1 Robustness............................................................................................................................45
6.1.2 How to interpret response....................................................................................................45
6.1.3 Choosing model....................................................................................................................46
6.1.4 Screening of mAb libraries...................................................................................................46
6.1.5 Limitations in Gyrolab Affinity Measurements.....................................................................47
6.1.6 Advantages of the Gyrolab for Affinity Measurements.........................................................47
7 CONCLUSIONS..................................................................................................................................49
8 FUTURE STUDIES............................................................................................................................49
9 ACKNOWLEDGEMENTS................................................................................................................51
10 REFERENCES....................................................................................................................................51
11 APPENDIX A: PROTOCOL FOR LABELLING OF IGG WITH ALEXA 647..........................53
12 APPENDIX B: PROTOCOL FOR LABELLING OF H-TSH WITH BIOTIN............................54
13 APPENDIX C: MATLAB CODE – ILLUSTRATING KD.............................................................55
14 APPENDIX D: MATLAB CODE – SOLVING KINETICS...........................................................57
15 APPENDIX E: MATLAB CODE – CURVE FIT OF KINETICS..................................................58