Monoclonal Antibodies

503 Pages·1992·24.68 MB·English

by  J. H. Peters

SPRINGER
LABORATORY
J. H. Peters H. Baumgarten (Eds.)
Monoclonal
Antibodies
With Contributions by Numerous Authors
Foreword by Georges Kohler
With 74 Figures
Springer-Verlag
Berlin Heidelberg New York London Paris
Tokyo Hong Kong Barcelona Budapest
Professor Dr. med. JOHANN HINRICH PETERS
Universitat G6ttingen
Abteilung fUr Immunologie
Kreuzbergring 57
W-3400 G6ttingen, FRG
Dr. rer. nat. HORST BAUMGARTEN
Forschungszentrum Tutzing
der Boehringer Mannheim GmbH
BahnhofstraBe 9-15
W-8l32 Tutzing, FRG
Translated by: Dr. PAUL DEBBAGE
Drawings and design by: Dr. rer. nat. ROBERT GIESELER
ISBN-13: 978-3-642-74534-8 e-ISBN-13: 978-3-642-74532-4
001: 10.1007/978-3-642-74532-4
Library of Congress Cataloging-in-Publication Data. Monoclonal antibodies / [edited by]
J. H. Peters, H. Baumgarten; with contrihutions hy numerous authors; foreword by Georges
Kohler. p. cm. -- (Springer lahoratory) Includes hibliographical references and index.
DM74. l. Mon9clonal anti-hodies--Lahoratorv manuals.
I. Peters, J. H. (Johann Hinrich) II. Baumgarten, H. (Horst)
III. Series. QRI86.85.M6566 1992 616.0T93--dc20 92-30163
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© Springer-Verlag Berlin Heidelberg 1992
Softcover reprint of the hardcover 15 t edition 1992
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Foreword
The present new version of this popular laboratory manual is at the
same time the first one of this text in the English language - and this
makes me even a little proud, as it reminds me of probably the first
collection of monoclonal recipes in English, written by myself, which
circulated for a couple of years in many laboratories.
In the meantime many researchers have put enormous effort into
improving methods for monoclonal antibody production. The proce
dures have become more and more standardized and by this have more
and more lost the character of magic secrets.
Hinrich Peters and Horst Baumgarten, who had followed this good
tradition already in the previous edition, written in German, suc
ceeded in making laboratory tricks teachable. They had contributed
their own experiences in cell culture and immunology, and were able to
engage a number of experienced authors to contribute to the work.
They were all willing to follow the general concept of this book, which
contains a brief theoretical background for the methods described and
presents the procedures in a highly organized structure. So the book
has retained its shape as a "cook-book", which I especially like.
The editors emphasize keeping the number of animals used as low
as possible, but as animals still have to be used in this technique, the
authors give advice for expert and professional handling, which may
be an important contribution to animal care. Producing human
monoclonal antibodies from human peripheral blood cells may be the
most straightforward development, also in reducing the use of
animals. Although these techniques are not yet as mature as the mouse
technology, the editors have succeeded in including a complete series
of methods for producing human antibodies.
Also, the editors have succeeded in eliminating methods using
radioactive tracers and replacing them by the most recent immunolog
ical methods.
Thus, I feel that this book has every chance of being acknowledged
as a standard collection of recipes, better suited to lie on the laboratory
bench rather than to rest on library shelves.
GEORGES KOHLER
Preface
This work started some years ago as a modest collection of photoco
pied laboratory recipes which were distributed to students of a
monoclonal antibody training course. Copies of the protocols multip
lied rapidly, and even spread to Eastern Germany, where labs bene
fited from them which otherwise had only little access to scientific
books. The first and second editions, although written in German,
even reached China and the USA, although we have never attempted
to re-establish German as the scientific world language.
As a consequence, the Springer Verlag persuaded us to publish the
new version in English.
The impetus for writing this text was our own experience that, for
instance, the clone yield obtained after fusion could be increased by
one to two orders of magnitude (!) if one carefully optimized each step
within the sequence of procedures leading to hybridomas. It was
therefore also our aim to ask our co-authors to follow some strict rules
in describing as briefly as possible the most efficient protocols for their
respective methods. The authors were kind enough to subject them
selves to these rigid rules and even accepted shortenings and interven
tions by the editors.
Thus, the user will be guided through this "cook-book" and will be
helped by frequent hints and even protocols for trouble shooting.
Although not presenting a complete handbook, we have focused on
those areas which usually cause most of the problems for newcomers,
i.e., immunization, cell culture, animal care, and mass production of
antibodies.
Animals are used and may still be necessary as donors of immune
cells for another decade; but human monoclonal antibodies are
strongly gaining terrain because of their different and wider specifici
ties. Both approaches are included in this manual: expert handling of
animals is described as well as the use of human peripheral blood cells
for generating hybridomas.
The reader will find a number of original descriptions not published
elsewhere to date, such as the reduction of endogenous peroxidase, in
vitro immunization, or the proper storage of cells.
It took us three editions to get rid of the last method still using
radioactive material-immunoprecipitation. It has been replaced by a
description of a very recent method based on biotinylation.
VIII Preface
Thanks to the help of many readers, some errors in the second
edition could be eliminated; thanks to our own efforts we may have
added some new ones. Therefore we would again like to ask the readers
to detect these errors and to win a "thank you" in a (possible) next
version.
Again, we have included product names and ordering numbers.
Such information will necessarily be incomplete. When naming a
substance or a supplier under the prefix "e.g.", we believe that
numerous others will perform equally well. The products named here
were simply those which were actually used when applying the
respective techniques - and were proven to work successfully.
We would like to thank many colleagues for manifold advice.
Especially we would like to thank Mrs. Dorothea Fey-Ostermeier,
Mrs. Rita Kuhn, and Mr. Detlef Friedrichs, who helped us with the
manuscript, and who have been our expert companions in the
laboratories. Ursula, Hanna, and Nils Peters have kindly read parts of
the manuscripts. Mrs. Ingrid Teuteberg and Mrs. Beatrice Ebert
(Anatomische Anstalt, UniversiUit Miinchen) typed the manuscript.
Thanks to all of them.
1. HINRICH PETERS
HORST BAUMGARTEN
Contents
1 Introduction .................................... 1
1.1 Principles of Cell Hybridization ................... 1
1.2 Properties and Significance of Monoclonal Antibodies. 4
1.3 Use of Monoclonal Antibodies in Human Beings:
Quality Control and Legal Aspects ................ 11
2 Preconditions for Hybridoma Technology ............ 18
2.1 Experimental Work with Animals ................. 18
2.1.1 Legal Aspects .................................. 18
2.1.2 Animal Maintenance ............................ 18
2.2 Equipment of the Cell Culture Laboratory .......... 30
2.3 Equipment for Immunological and Biochemical Work. 36
2.4 Organization of the Course of Work (Time Table)
and Estimation of Costs .......................... 37
3 Immunization ................................... 39
3.1 Principles and Strategies for Immunizing Animals ... 39
3.2 Choice of the Immunogen ........................ 40
3.2.1 Native Antigens ................................. 40
3.2.2 Modified or Synthetic Antigens ................... 42
3.3 Immunizing the Larger Experimental Animals
for Antisera Production .......................... 45
3.4 Immunizing Mice ............................... 47
3.4.1 The Basics of Immunizing Mice
for Hybridoma Production ....................... 47
3.4.2 Methods of Immunizing Mice ..................... 50
3.5 Influencing the Immune Response ................. 57
3.5.1 Influencing the Immune Response
by Use of Selected Mouse Strains .................. 57
3.5.2 Influencing the Immune Response
by Use of Adjuvants ............................. 58
3.5.3 Influencing the Immune Response
by Inducing Tolerance ........................... 63
3.5.4 Modifying the Immune Response by Use of Cytostatica 67
3.5.5 Modulating the Immune Response by Masking
Especially Immunogenic Epitopes with Antibodies ... 68
3.5.6 Modifying the Immune Response
to Generate Certain Immunoglobulin Subclasses ..... 69
X Contents
4 Taking Blood and Isolating Cells ................... 71
4.1 Taking Blood from Experimental Animals .......... 71
4.1.1 Taking Blood from Mice ......................... 71
4.1.2 Taking Blood from Rats ......................... 74
4.1.3 Taking Blood from Rabbits. . . . . . . . . . . . . . . . . . . . . . . 75
4.1.4 Taking Blood from Sheep and Goats ............... 77
4.2 Isolating Lymphocytes from Spleen and Lymph Nodes 78
4.3 Isolating Human Lymphocytes from Peripheral Blood,
Tonsils, or Spleen ............................... 80
4.4 Enriching Antigen-Specific Lymphoblasts for Fusion. 82
4.5 Isolating Mouse Peritoneal Macrophages
for Use as Feeder Cells ........................... 86
5 Cell Culture .................................... 88
5.1 Requirements for Cell Culture .................... 88
5.1.1 Cleaning, Disinfecting, and Avoiding Toxicity ....... 88
5.1.2 Plastic Ware, Water, Media, Sera, and Additives ..... 91
5.1.3 Culture Conditions .............................. 95
5.2 Additives to Media:
Growth Factors, Conditioned Media ............... 98
5.3 Cryopreservation of Cells ........................ 10 1
5.3.1 Freeze Storage of Cells Directly After Fusion ....... 101
5.3.2 Freeze Storage of Hybridomas in Cell Culture Plates . 106
'5.3.3 Storing Lymphocytes in the Cold .................. 108
5.3.4 Keeping Track of Frozen Cells by Use of Computers . 109
5.4 Bacterial and Fungal Infections ................... 110
5.5 Limiting an Infection in Multi-Well Plates .......... 113
5.6 Mycoplasmas ................................... 114
5.6.1 Mycoplasma Enrichment Cultures in Cell-Free Media. 116
5.6.2 Fluorescence Test to Demonstrate Mycoplasma
Infections in Cultures of Adherent or Suspended Cells. 122
5.6.3 Immunological and Genetical Tests for Mycoplasmas. 128
5.6.4 Cleaning Mycoplasma-Infected Cells ............... 129
5.6.4.1 Use of Antibiotics to Eliminate Mycoplasmas ....... 129
5.6.4.2 Clearing Mycoplasmas from Infected Cells
by Co-Culture with Macrophages .................. 130
5.7 Cell Viability Testing Using Fluorescent Dyes ....... 133
6 Production of Hybridomas ........................ 137
6.1 Basics ......................................... 137
6.1.1 Properties and Production of Myeloma
and Tumor Cell Lines ........................... 137
6.1.2 Principles of Selection ........................... 139
6.1.3 Survey of Mouse Myelomas ...................... 148
6.2 Fusing Cells to Generate Mouse
Monoclonal Antibodies .......................... 149
Contents XI
6.3 Human Hybridoma Technique .................... 157
6.3.1 Fusion Partner Cell Lines and Methods
for Generating Human Monoclonal Antibodies ...... 157
6.3.2 Basics of in Vitro Immunization ................... 164
6.3.3 Preparation of Human B-Lymphocytes
for in Vitro Immunization: Principles .............. 170
6.3.4 Preparing the Cells .............................. 171
6.3.4.1 Removal ofT-Lymphocytes by Panning ............ 171
6.3.4.2 Harvesting Monocytes by Adherence .............. 172
6.3.4.3 Differentiating Out Accessory Cells from Monocytes 172
6.3.4.4 Removal of Lysosome-Rich Cells .................. 174
6.3.5 In Vitro Immunization: Additives ................. 175
6.3.5.1 T-Cell Rosetting and Preparing
a Conditioned Medium .......................... 176
6.3.6 Procedures for in Vitro Immunization
of Human Lymphocytes ......................... 177
6.3.7 Fusion of Human Cells .......................... 180
6.3.8 Epstein-Barr Virus (EBV) Transformation
and EBV Hybridoma Technique .................. 181
6.3.8.1 EBV Transformation ............................ 181
6.3.8.2 Heteromyeloma Technique:
Fusion of EBV-Transformed B-Lymphocytes
with Mouse Myeloma Cells ....................... 184
6.3.8.3 Transfecting the Geneticin-Resistance Gene ......... 185
6.3.9 Fusion with Cytoplasts .......................... 186
6.3.10 DNA Transformation ........................... 188
6.4 Other Fusion Methods ........................... 190
6.4.1 Fusion with Viruses ............................. 190
6.4.2 Electrofusion ................................... 190
6.5 Calculating the Number
of Hybridoma Clones To Be Expected ............ . 192
6.6 Culture and Enrichment of Hybridomas ........... . 194
6.6.1 Growing Hybridomas, Trial Plating .............. . 194
6.6.2 Trouble-Shooting During the Generation
and Culturing of Hybridomas .................... . 197
6.7 Cloning Cells .................................. . 202
6.7.1 Limiting-Dilution Cloning ....................... . 203
6.7.2 Cloning Cells with a Cell Sorter .................. . 206
6.8 Identifying Human Genomic Material
in Mouse-Human Hybridomas ................... . 211
6.9 Fine-Tuning Hybridomas ....................... . 211
6.9.1 Increasing the Proportion of Hybridomas Specific
for the Desired Antigen ......................... . 211
6.9.2 Class-Switch Variants ........................... . 213
6.9.3 Strategies for Generating Stable Hybridomas
Producing Human Monoclonal Antibodies ......... 215
XII Contents
6.9.4 Bispecific, Chimeric, and Recombinant Antibodies 217
6.10 Nomenclature of Monoclonal Antibodies ........... 220
7 Mass Production of Monoclonal Antibodies .......... 223
7.1 Mass Production of Monoclonal Antibodies
in Cell Culture or Ascites ......................... 223
7.2 Production of Monoclonal Antibodies in Mice ...... 225
7.2.1 Production of Murine Monoclonal Antibodies
in the Peritoneal Cavity of the Mouse .............. 225
7.2.2 Production of Human Monoclonal Antibodies
in the Peritoneal Cavity of the Mouse .............. 233
7.3 Production of Monoclonal Antibodies
in Bioreactors ................................. . 235
7.4 Serum-Free Cell Culture ........................ . 245
7.5 Checking the Antibody Properties ................ . 252
7.5.1 Proto colling the Production and Quality Control
of Monoclonal Antibodies ....................... . 252
7.5.2 Genome Stability of Mouse Hybridomas 254
8 Purifying Monoclonal Antibodies
and Producing Antibody Fragments ................. 258
8.1 Purification of Monoclonal Antibodies: an Overview 258
8.1.1 Ammonium Sulfate Precipitation of Monoclonal
Antibodies from Hybridoma Ascites Fluid .......... 261
8.1.2 Protein A/Protein G Column Chromatography ...... 264
8.1.3 Anion Exchange Chromatography for Purification
of Monoclonal IgG Antibodies .................... 271
8.2 Producing Immunoreactive Fragments
from Mouse Monoclonal Antibodies ............... 275
8.2.1 Preparing Fab Fragments ........................ 277
8.2.2 Preparing F(ab)2 Fragments ...................... 281
9 Coupling Monoclonal Antibodies ................... 285
9.1 Basic Principles ................................. 285
9.2 Conjugation of Enzymes to Monoclonal Antibodies .. 292
9.2.1 Conjugating Peroxidase .......................... 294
9.2.2 Conjugating ~-Galactosidase ..................... 296
9.2.3 Conjugating Alkaline Phosphatase ................. 298
9.3 Biotinylating Monoclonal Antibodies .............. 299
9.4 Conjugating Fluorochromes to Monoclonal Antibodies 303
9.4.1 FITC Conjugation .............................. 303
9.4.2 Conjugation of Rhodamine, Phycoerythrin,
and Cyanine Dyes ............................... 306
9.5 Conjugating Monoclonal Antibodies
to Solid Phases (Immune Absorption) .............. 307