Table Of ContentCambridge Studies in Biotechnology
Editors: Sir James Baddiley, N. H. Carey, I. J. Higgins, W. G. Potter
8 Monoclonal antibodies in biology and biotechnology:
theoretical and practical aspects
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Monoclonal antibodies in biology
and biotechnology: theoretical and
practical aspects
KENNETH C. McCULLOUGH
Institut fur Viruskrankheiten und Immunprophylaxe
Basle, Switzerland
and
RAYMOND E. SPIER
Department of Microbiology, The University of Surrey
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© Cambridge University Press 1990
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First published 1990
A catalogue record for this publication is available from the British Library
Library of Congress Cataloguing in Publication data
McCullough, Kenneth C. (Kenneth Charles), 1951-
Monoclonal antibodies in biology and biotechnology : theoretical and practical
aspects / Kenneth C. McCullough and R.E. Spier.
p. cm. — (Cambridge studies in biotechnology : 8)
Bibliography: p.
Includes index.
ISBN 0 521 25890 1
I. Antibodies, Monoclonal—Biotechnology. I. Spier, R. (Raymond)
II. Title. III. Series.
TP248.65.M65M33 1990
660'.63—dcl9 89-914 CIP
ISBN 978-0-521-25890-6 hardback
Transferred to digital printing 2008
To our parents and our wives, without whom we never
would have had a hot meal.
Vll
Contents
Page
1 Introduction: the immune response 1
1.1. Introduction 1
1.2. A history of the early development of immunology 1
1.3. The antibodies 5
1.3.1. Antibody production: response to antigen 17
1.3.2. Other immunological processes 21
1.4. The immune response 21
1.4.1. What is 'specificity'? 24
1.4.2. Non-specific defence mechanisms 27
1.4.3. Specific reactions 29
1.4.4. Antigen-processing cells, APC 30
1.4.5. Regulatory networks 44
1.4.6. The B lymphocyte and antibody production 52
1.4.7. Antibody-dependent cellular cytotoxicity
reactions (ADCC) 56
1.4.8. Antibody-independent cell-effector mechanisms 61
1.4.9. Memory 63
1.4.10. Concluding remarks 64
1.5. Affinity of antibodies and the immune response 64
1.5.1. Affinity of Monoclonal antibodies (MAb) and
its relevance to the avidity of antiserum 65
1.5.2. Affinity of the immune response and the
relevance to memory 67
1.6. Antibody response 70
1.6.1. Cellular interaction 71
1.6.2. Lymphocyte receptors and antibody secretion 72
1.6.3. Antibody production and its relevance to
the generation of hybridomas 76
1.6.4. The relevance of antibody classes to the
immune response 78
1.6.5. Antibody isotype distribution in body fluids 81
1.6.6. Antibody-mediated anaphylacticreaction 82
1.6.7. The humoral immune response: summary 85
1.7. Monoclonal antibodies (MAb) 86
1.8. Summary 87
viii Contents
Page
2 Making hybridomas (hybridoma technology) 90
2.1. Basic requirements 90
2.2. The myeloma cell 92
2.2.1. Definition 92
2.2.2. Myeloma cell induction 92
2.2.3. Detection 92
2.2.4. Characteristics 94
2.3. The production of hybridomas from myeloma cells 96
2.3.1. Fusion between splenocytes and myeloma cells 96
2.3.2. Selective procedures 99
2.4. Myeloma cells available 102
2.5. Generation of hybridoma cell lines 108
2.5.1. Choice of animal for immunisation 109
2.5.2. Choice of immunisation protocol 109
2.5.3. Choice of immunopotentiating agents 113
2.5.4. The final inoculation 114
2.5.5. Removal of immune cells 116
2.5.6. Which lymphocytes produce the best hybridoma? 118
2.5.7. From splenocyte to hybridoma: the alternative
methods 121
2.5.8. Choice of myeloma cells 123
2.5.9. The proposed genetic requirements for
hybridoma stability 124
2.5.10. The use of genetically related myeloma cells
and splenocytes 124
2.6. A practical guide to generating hybridoma cell lines 126
2.6.1. Preparation of lymphocytes 126
2.6.2. Fusion 128
2.6.3. Selection of hybridomas (Fig 2.15) 130
2.7. Types of hybridoma cell line 133
2.7.1. Generation of cell hybridomas 134
2.8. Summary 136
3 Factors affecting successful hybridoma
production 139
3.1. Efficacy of immunisation 140
3.2. Source of myeloma cells 149
3.3. Source of PEG 149
3.4. Fusion ratio 150
3.5. Source of media and media supplements 152
3.5.1. Comparisons of different sources of the medium 152
3.5.2. Medium supplements 156
3.6. Physical factors 167
Contents ix
Page
3.7. Growth factors 167
3.7.1. Feeder cells as sources of growth factors 169
3.7.2. Splenocyte feeder layers 170
3.7.3. Peritoneal exudate cell (PEC) feeder layers 170
3.7.4. Endothelial cell feeder layers 172
3.7.5. Fibroblastic (3T3/B and3T3/A31)
cell feeder layers 175
3.8. Culturing protocols 177
3.8.1. Initial growth of hybridomas after fusion 178
3.8.2. Subcultures of hybridomas 180
3.8.3. Removal of aminopterin 183
3.8.4. Screening for antibody production,
and cloning of cells 183
3.9. Summary 183
4 Selection of monoclonal antibody-secreting
hybridoma cell lines 185
4.1. Screening of polyclonal hybridomas 186
4.1.1. Enzyme-linked immunosorbent assay (ELISA) 186
4.1.2. Radioimmunoassay (RIA) 198
4.1.3. Passivehaemagglutination (PHA) 198
4.1.4. OuchterlonyDID 208
4.1.5. Single radial immunodiffusion (SRID) 211
4.1.6. Radial haemolysis (RH) 214
4.1.7. Immunoelectrophoresis (IEP) 217
4.1.8. Haemadsorption (rosette formation) 224
4.1.9. Direct anti-globulin rosette reaction (DARR) 226
4.1.10. Indirect anti-globulin rosette reaction (IARR) 226
4.1.11. Haemolytic plaque assay (HPA)orPFC assay 228
4.1.12. Replica plate assay (RPA) 228
4.1.13. Haemagglutination inhibition (HAI) 230
4.1.14. Haemolysin inhibition (HLI) 231
4.1.15. Neutralisation 231
4.1.16. Opsonisation 232
4.1.17. Immunofluorescence 232
4.1.18. In vitro lymphocyte assays 234
4.1.19. Other assays 236
4.1.20. Assay of MAb: summary 240
4.2. Cloning 242
4.2.1. Isolation of colonies from 'masterplates' 243
4.2.2. Cloning by limit dilution 244
4.2.3. Cloning in semi-solid medium 247
4.2.4. Micromanipulation 254
Contents
Page
4.3. Cloning immediately after fusion 257
4.3.1. Cloning with pre-formed feeder cells 258
4.3.2. Cloning with adjacent feeder cells 258
4.3.3. Cloning using a feeder cell-hybridoma
'monolayer' 258
4.3.4. The 'fusion/cloning' procedure 259
4.4. Determination of monoclonality 261
4.5. Culturing of monoclonal antibody-secreting hybridoma
cell lines 263
4.6. Summary 264
5 The large-scale production of monoclonal
antibodies in vitro 265
5.1. Introduction 265
5.2. The cell population 267
5.3. Culture media 268
5.3.1. Hybridoma isolation media 270
5.3.2. Hybridoma establishment media 271
5.3.3. Intermediate phase media 271
5.3.4. Serum-free media 273
5.3.5. Alternative approaches to medium formulation 274
5.3.6. Relationship between cell growth and antibody
production 274
5.3.7. Culture techniques for the large-scale
production of monoclonal antibodies from
hybridoma cell lines 275
5.3.8. Overview of present techniques 278
5.3.9. Environments surrounding animal cells in vivo 279
5.4. A survey of the advantages and disadvantages of
techniques based on dense cell systems 285
5.4.1. What are dense cell cultures? 285
5.4.2. Some advantages of dense cell cultures 285
5.4.3. Some disadvantages of dense cell cultures 288
5.5. The influence of controllable parameters on
monoclonal antibody production by hybridoma cells
in culture 290
5.5.1. Physical factors 290
5.5.2. Nutritional factors 292
5.6. The technologies used to produce large quantities of
monoclonal antibodies from cells held in culture 295
5.6.1. The scope of the problem 296
5.6.2. Cells in suspension 298
