ANTIBODY AFFINITY AND AFFINITY DISTRIBUTIONS DETERMINATION IN ELISA AND BIOSENSOR CIP-GEGEVENS KONINKLlJKE BIBLIOTHEEK, DEN HAAG Pathak, Sulbha Sanjeev Antibody affinity and affinity distributions. Determination in ELISA and biosensor/ Sulbha Sanjeev Pathak; [ill.: Tar M. van Osl. - Rotterdam: Erasmus Universiteit, Afdeling Immunologie. - III. Thesis Rotterdam. - With ref. ISBN 90-73436-27-3 NUGI743 Subject headings: antibodies; affinity / enzym immunoassay/ biosensor No part of this thesis may be reproduced or transmitted in any form by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system, without permission in writing from the publisher (S.S. Pathak, Department of Immunology, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands). ANTIBODY AFFINITY AND AFFINITY DISTRIBUTIONS DETERMINATION IN ELISA AND BIOSENSOR ANTISTOF AFFINITEIT EN AFFINITEITSDISTRIBUTIES BEPALING IN ELISA EN BIOSENSOR PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof. Dr. P.W.C. Akkermans M. A. en volgens besluit van het College voor Promoties. De open bare verdediging zal plaatsvinden op woensdag 20 december 1995 om 11.45 uur. door Sulbha Sanjeev Pathak geboren te Sang Ii, India PROMOTIE-COMMISSIE: Promotor: Prof. dr. R. Benner Co-promotor: dr. H.F.J. Savelkoul Overige leden: Prof. dr. H.G. van Eijk Prof. dr. J.J. Haaijman Prof. dr. H.J. Neijens IMMUNOLOGIE The studies described in this thesis were performed at the ROTTERDAM Department of Immunology, Erasmus University Rotterdam. Publication of this thesis was partly sponsored by Fisons Applied Sensor Technology, Cambridge, UK Illustrations: Tar M. van Os Printing: Ridderprint, Ridderkerk To raise new questions, new possibilities, to regard old problems from a new angle makes a real adventure in science. Where change is the only constant (Einstein) To Sanjeev and Gauri Contents CHAPTER 1 Introduction 9 CHAPTER 2 Determination of concentration and affinity in ELISA 45 2.1 Quantitation of immunoglobulin concentration in ELISA 47 Manual of Immunological Methods. I. Lefkovits (Ed), in press 2.2 Terasaki-ELISA for murine IgE. II. Quantitation of absolute concentration of antigen-specific and total IgE 75 J. Immunol. Methods 1989; 116:277-285 2.3 Terasaki-ELISA for murine IgE. III. Determination of absolute concentration and functional affinity by sequential equilibrium binding analysis 85 J. Immunol. Methods 1989;183:71-81. 2.4 Mixing of two monoclonal Ig E antibodies increases the functional affinity of the mixture for the antigen 97 Submitted 2.5 Determination of relative affinity and affinity distributions in ELISA 107 Submitted CHAPTER 3 Kinetic interaction analysis with a biosensor 115 3.1 Determination of antibody affinity and affinity distributions 117 Manual of Immunological Methods. I. Lefkovits (Ed), in press 3.2 Application of a resonant mirror biosensor to the determination of functional affinity of monoclonal and polyclonal antibodies 141 Submitted 3.3 Interleukin-4 increases the pace of affinity maturation of IgG 1 antibodies 159 Submitted CHAPTER 4 Discussion 175 SUMMARY 187 SAMENVA TTING 191 ABBREVIATIONS 195 GLOSSARY 197 DANKWOORD 199 CURRICULUM VITAE 201 PUBLICATIONS 202 INTRODUCTION Introduction 1.1 Humoral immunity: the role of B cells 1.2 Helper T cells in humoral immunity 1.3 Specificity and diversity 1.3.1 The V(D)J recombination 1.3.2 Somatic hypermutation 1.3.3 Class switch recombination 1.4 Memory 1.4.1 Phenotype of memory Band T cells 1.4.2 Mechanisms of immunological memory 1.5 The germinal center 1.6 The aim of the experimental work 1 .7 References 'l?tl 31 'l11 ~ G,'~ 00 ~ "ll ~1'fl: