Table Of ContentANTIBODY TECHNOLOGY
The INTRODUCTION TO BIOTECHNIQUES series
Editors:
J.M. Graham Merseyside Innovation Centre, 131 Mount Pleasant,
Liverpool L3 5TF
D. Billington School of Biomolecular Sciences, Liverpool John
Moores University, Byrom Street, Liverpool L3 3AF
Series adviser:
P.M. Gilmarlin Centre for Plant Biochemistry and Biotechnology,
University of Leeds, Leeds LS2 9JT
CENTRIFUGATION
RADIOISOTOPES
LIGHT MICROSCOPY
ANIMAL CELL CULTURE
GEL ELECTROPHORESIS: PROTEINS
PCR
MICROBIAL CULTURE
ANTIBODY TECHNOLOGY
Forthcoming titles
GENE TECHNOLOGY
LIPID ANALYSIS
GEL ELECTROPHORESIS: NUCLEIC ACIDS
PLANT CELL CULTURE
LIGHT SPECTROSCOPY
MEMBRANE ANALYSIS
ANTIBODY TECHNOLOGY
Eryl Liddell
School of Molecular and Medical Biosciences, Museum Avenue,
PO Box 911, Cardiff CFl 3US, UK
lan Weeks
Department of Medical Biochemistry, University of Wales College of
Medicine, Heath Park, Cardiff CF4 4XN, UK
{1IOS
SCIENTIFIC
PUBLISHERS
© BIOS Scientific Publishers Limited, 1995
First published 1995
Transferred to Digital Printing 2005
All rights
All rights reserved. No part of this book may be reproduced or transmitted,
in any form or by any means, without permission.
A CIP catalogue record for this book is available from the British Library.
ISBN 1 872748 87 2
BIOS Scientific Publishers Ltd
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Typeset by Chandos Electronic Publishing, Stanton Harcourt, UK.
Contents
Abbreviations ix
Preface xi
PART 1: BASIC PRINCIPLES AND METHODS
1. The Immune System 1
Introduction 1
Antibodies 2
Antibody biosynthesis 4
2. Production of Polyclonal Antibodies 9
Manipulation of the immune response 9
Antibody production 10
Mode of immunization 10
Dose of immunogen 11
Timing of immunizations 11
Characterization of antibodies 12
Titer 12
Affinity 15
Cross-reactivity 18
Immunoglobulin class 19
Chemical manipulations 20
Hapten-carrier protein conjugates 20
Solid-phase reagents 21
Labeled reagents 23
References 23
3. Production of Monoclonal Antibodies 25
Introduction 25
Basic methodology 26
Immunization 26
Tissue culture 28
Myelomas 28
Fusion 30
HAT selection 31
Antibody screening tests 32
v
vi ANTIBODY TECHNOLOGY
Cloning 33
Storage (cryopreservation) 35
Propagation of clones 35
Human hybridomas 38
Variations on basic methodology of production 38
Alternative lymphocytes 38
In vitro immunization 39
Heteromyelomas and hybrid hybridomas 40
Electrofusion 41
Alternative cloning methods 41
Characterization of antibodies 42
Making or buying: relative merits 42
References 44
4. Antibody Production by Chemical and Genetic
Engineering 45
Introduction 45
Chemical modification 46
Antibody fragmentation: Fab and F(ab')2 46
Bispecific antibodies 48
Altering effector functions 49
Genetic manipulation 50
Humanizing rodent antibodies 50
Combinatorial antibody gene libraries 52
Summary and future prospects 62
References 63
PART 2: TECHNIQUES AND APPLICATIONS
5. Diagnostic Applications of Antibodies 65
Immunoprecipitation reactions 65
Radioimmunoassay 68
Radioactive labeling 69
Separation systems 71
Immunoradiometric assays 72
Other immunoassay architectures 74
Nonisotopic immunoassays 75
Enzyme immunoassay 76
Fluorescence immunoassay 78
Chemiluminescence immunoassay 79
Homogeneous immunoassays 81
Summary 82
References 83
CONTENTS vii
6. Immunolocalization 85
Introduction 85
Immunocytochemistry 85
Tissue and cell preparation 86
Choice of antibody 88
Choice of antibody label 89
Labeling strategies 94
Double and triple labeling 96
Nonspecific staining 99
Immunoblotting 100
References 102
7. Therapeutic Applications of Antibodies 103
Introduction 103
Antibody type and specificity 104
Polyclonal antisera 104
Rodent monoclonal antibodies 105
Humanized and CDR-grafted antibodies 110
Recombinant antibodies 111
Effector functions 111
Unlabeled antibodies 111
Labeled antibodies 112
Regulatory issues 117
Future prospects 118
References 118
8. Other Applications of Antibodies 121
Immunopurification 121
Biosensors 124
Catalytic antibodies 126
Epitope mapping 127
Fluorescence-activated cell sorting (FACS) 129
References 129
Appendices
Appendix A: Glossary 131
Appendix B: Suppliers 135
Appendix C: Further reading 140
Index 143
Abbreviations
8-AG 8-azaguanine
ADCC antibody-dependent cell-mediated cytotoxicity
ADEPT antibody-directed enzyme prodrug therapy
AP alkaline phosphatase
APAAP alkaline phosphatase-anti-alkaline phosphatase
C constant
CDR complementarity determining regions
CEA carcinoembryonic antigen
CG chorionic gonadotropin
CHO Chinese hamster ovary
DAB 3.3'-diaminobenzidine
dAb single-domain antibody
EBV Epstein-Barr virus
EF-2 elongation factor 2
ELISA enzyme-linked immunosorbent assay
EMIT enzyme-multiplied immunoassay technique
FACS fluorescence-activated cell sorter
FCA Freund's complete adjuvant
FIA Freund's incomplete adjuvant
FITC fluorescein isothiocyanate
FSH follicle-stimulating hormone
GVDH graft-versus-host disease
HAMA human anti-mouse antibody
HAT hypoxanthine-aminopterin-thymidine
HGPRT hypoxanthine guanine phosphoribosyl transferase
HIV human immunodeficiency virus
HRP horseradish peroxidase
id idiotypic
Ig immunoglobulin
IL interleukin
IRMA immunoradiometric assay
IVIG intravenous immunoglobulin
J joining
LH luteinizing hormone
MHC major histocompatibility complex
MPO microperoxidase
NK natural killer
PAP peroxidase-antiperoxidase
PCR polymerase chain reaction
PEG polyethylene glycol
RIA radioimmunoassay
ix
