Department of Immunology,. Erasmus University Rotterdam. Publication of this thesis was partly sponsored by Fisons Applied Sensor. Technology, Cambridge ...
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 ROTTERDAM
The studies described in this thesis were performed at the Department of Immunology, Erasmus University Rotterdam.
Publication of this thesis was partly sponsored by Fisons Applied Sensor Technology, Cambridge, UK Illustrations: Printing:
Tar M. van Os 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
CHAPTER 2
Determination of concentration and affinity in ELISA
45
Quantitation of immunoglobulin concentration in ELISA
47
2.1
9
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 3.1
Kinetic interaction analysis with a biosensor
115
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 1.4.2
Phenotype of memory Band T cells Mechanisms of immunological memory
1.5 The germinal center 1.6 The aim of the experimental work 1 .7 References
31 'l11 'l?tl ~ G,'~
00 ~ "ll ~1'fl:
11 CHAPTER 1 Introduction Neither the horse, nor the elephant, definitely no\ the tiger, but the weak and defenceless lamb is chosen for sacrifice - states a sanskrit observation coined about 1500 years ago (adjacent page). The ability to defend oneself against external and internal aggressors is indispensable for survival. Some form of selfdefence is a basic requirement needed to protect oneself from a world of pathogens that is forever evolving. In mammals, the body's defence system is highly evolved. In them, the non-specific innate immune system and the specific adaptive immune system are linked together to form a formidable integrated host defence mechanism.
1.1 Humoral immunity: the role of B cells In mammalian adults, bone marrow is the major site of the generation of B lymphocytes ll94. 1951. The antigen-independent phase of B-cell maturation also takes place in the bone marrow, where lymphoid progenitors commit to the B cell lineage and subsequently differentiate into surface immunoglobulin M positive (lgM +) B cellsl20BI. The differentiation of B lymphocytes from committed precursor cells to 19 secreting plasma cells is a multiple-step procedure that can be defined by changes in the expression pattern of lineage-specific genes l9B• 1521. Mature B cells exit from the bone marrow and migrate to peripheral lymph nodes and other peripheral lymphoid organs, where the antigen-dependent phase of development takes place if the B cells encounter their specific antigen l1461 . The terminal differentiation of B cells to antibody-secreting plasma cells takes place in these organs l9BI . These plasma cells are end cells with the sale function of producing antibodies - the effectors of humoral immunity.
1.2 Helper T cells in humoral immunity T lymphocytes are also produced by the bone marrow stem cells, but mature in the thymus. CD4 + T cells regulate the humoral immune response by selectively inducing antigen-specific B cells to differentiate and secrete antibodies with different effector functions determined by the various Ig heavy chain isotypesl1491. Antibody responses to protein antigens require specific recognition of the same antigen complex by both B lymphocytes and CD4 + T lymphocytes. Such antigens are called T dependent (TO) antigens and the T cells involved are referred to as T helper (Th) cells l27 7). Protein antigens are processed by antigen-presenting cells (APC) and presented to Th cells in the context of MHC class 11 molecules along with costimulatory signals. The APC also produce IL-1. Together, these two events activate the T cells. Cross-linking of antigen receptors on cell surface along with help from activated Th cells activates the B cells. B cells can also obtain help directly from T cells by acting
12
CHAPTER 1
as antigen-specific APC, T cell help has two components - release of cytokines and additional signals which require cell to cell contact, i.e, cognate help'
