Monoclonal Antibodies: A Practical Approach

514 Pages·2000·52.75 MB·English

by  Shepherd P.

Monoclonal Antibodies
The Practical Approach Series
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* indicates a forthcoming title
Please see the Practical Approach series website at
http://www.oup.co.uk/pas
for full contents lists of all Practical Approach titles.
Monoclonal Antibodies
A Practical Approach
Edited by
Philip Shepherd
Guy's, King's and St Thomas' Schools of
Medicine, Peter Gorer Department of
Immunobiology, Guy's Hospital, New Guy's
House, London Bridge, London SE1 9RT, U.K.
and
Christopher Dean
Section of Immunology, Institute of Cancer
Research, McElwain Laboratories, 15 Cotswold
Road, Belmont, Sutton, Surrey SM2 5NG, U.K.
OXPORD
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British Library Cataloguing in Publication Data
Data available
Library of Congress Cataloging in Publication Data
Monoclonal antibodies : a practical approach / edited by Philip S. Shepherd,
Christopher J. Dean,
p.; cm.—(The practical approach series)
Includes bibliographical references and index.
ISBN 0-19-963723-7 (hbk.: alk. paper)—ISBN 0-19-963722-9 (pbk.: alk.
paper)
1. Monoclonal antibodies—Laboratory manuals. I. Shepherd, Philip S.
II. Dean, Christopher]. III. Series.
[DNLM: 1. Antibodies, Monoclonal. 2. Immunologic Techniques. QW
575.5A6 M7455 2000]
OJU86.85 M65646 2000 616.07'98—dc21 99-059426
135 79108642
ISBN 0-19-963723-7 (Hbk.)
ISBN 0-19-963722-9 (Pbk.)
Typeset in Swift by Footnote Graphics, Warminster, Wilts
Printed in Great Britain on acid-free paper
by The Bath Press (Avon) Ltd
Preface
This volume presented in the Practical Approach Series deals with the prep-
aration, testing and derivation of monoclonal antibodies as well as with some of
their applications. As with the previous book on antibodies in this series, edited
by David Catty, we have tried to maintain a similar format in making it a
collection of bench protocols, even if some of the techniques require specialised
apparatus not normally found in a routine molecular immunology laboratory.
The first part includes chapters dealing with the preparation of rodent and
human monoclonal antibodies by the standard somatic hybridisation technique,
as well as by recombinant techniques, including the use of phage libraries and
ARM display. Leading on from their preparation there are chapters dealing with
small and large scale production, including production in plants together with
purification techniques and methods for labelling them with radionuclides and
non-radioisotopic compounds such as enzymes or fluorochromes and colloidal
gold for light and electron microscopy. The second half covers their application,
with chapters on immunoblotting, enzyme linked immunoassays, immuno-
fluorescence, immunocytochemical staining of cells and FACS analysis. Also
included are chapters on the clinical applications of monoclonal antibodies in
rheumatoid arthritis, tissue typing and the management of organ graft rejection,
the detection of chemically modified DNA in lymphocytes from patients
undergoing chemotherapy and analysis of clinical haematological samples in
transplantation for malignancy.
New immunological techniques incorporating tried and tested methodol-
ogies are of interest to established Immunologists as well as those entering the
field for the first time. One or two of the chapters will be of interest to more
specialised workers, e.g. those using confocal microscopy, but still of interest to
those investigators whose laboratories lack such specialised apparatus.
The authors have been given a free rein to cover their topic in the manner
they chose. This has led to some repetition which we as editors have not
removed so as to keep the understanding and continuity within and between
chapters such that the reader does not have to keep referring to the other
chapters to follow the subject under presentation.
We are deeply indebted to all the authors for their hard work and dedication
v
in writing the chapters, and to Dora Paterson and Jeremy Cridland for their
secretarial and computer assistance.
Both of us have learnt a lot about new antibody production techniques while
editing this book and we hope this will be the same experience for all whom
refer to the finished volume.
P. Shepherd and C. Dean
Contentshh
List of protocols page xix
Abbreviations xxvii
1 Preparation of rodent monoclonal antibodies by In vitro somatic
hybridization 1
Christopher Dean and Philip Shepherd
1 Introduction and strategy 1
2 Choice of host for immunization and myeloma for cell fusion 3
3 Choice of immunogen 4
4 Preparation of antigen for immunization 5
Soluble antigens 5
Antigens expressed on live cells 5
Plasmid DNA 6
Peptides 7
5 Route of immunization 7
Generation of immune spleen cells 7
Immunization via the Peyer's patches of rats 7
6 Growth of myeloma cell lines 9
7 Preparation of cells for fusion 10
8 Cell fusion 11
9 Screening hybridoma culture supernatants for specific antibody 12
Antigen coated multiwell plates 12
Live or fixed cells 16
Ligand binding assays 17
10 Cloning of hybridomas 18
11 Characterization and use of the antibodies obtained 19
Isotyping of antibodies 20
Epitope reactivity 20
Specific immunoprecipitation 21
References 23
vii
CONTENTS
2 Preparation of recomblnant antibodies from immune rodent
spleens and the design of their humanization by CDR grafting 25
Olivier]. P. Leger and Jose W. Saldanha
1 Introduction 25
2 Preparation of mouse spleen 26
3 Isolation of total RNA from spleen 28
4 Poly(A+) mRNA isolation 30
5 Reverse transcriptase reaction 31
6 Primary PCR of antibody genes 33
7 Preparation of linker 37
8 Assembly of VH and VK gene fragments with linker DNA 39
9 Assembly of single chain Fv antibody fragments 41
10 Reamplification of assembled scFv DNA 43
11 Restriction enzyme digestion of assembled scFv 44
12 Purification of pHEN-1 vector by equilibrium centrifugation in CsCl
ethidium bromide gradients 46
13 Restriction digestion of the phage display vector, pHEN-1 49
14 Ligation of pHEN-1 and insert antibody scFv 50
15 Preparation of electroporation competent B. coli TGI strain cells 52
16 Electroporation 53
17 Analysis of recombinant clones from the library 55
References 57
Appendix 58
1 The design of the humanized antibody 58
Issues to consider 59
References 64
3 Selection of antibodies from phage libraries of immunoglobulin
genes 67
jane K. Osbourn
1 Introduction 67
2 Preparation and storage of phage library stocks 68
Phagemid libraries 68
Phage libraries 69
3 Maintenance of bacterial stocks and titration of phage preparations 69
4 Selection of phage libraries on purified, immobilized antigen 71
Immobilization of antigen on immunotubes 71
Elution conditions 72
Storage and rescue of the phagemid population after selection 72
Choice of number of selection rounds 73
5 Selection of phage libraries on biotinylated antigen 74
Biotinylation of antigen 74
Selections using biotinylated antigen 75
viii
CONTENTS
6 Cell surface selections 76
Adherent cell selections 77
Cells in suspension 78
Screening the output of cell surface selections 78
7 Proximity selections 78
Proximity selection using an existing antibody 80
Proximity selection using natural ligands 81
Step back selections 83
8 Screening of selected phage 83
Basic screening assays 83
Affinity screening 87
9 Soluble scFv production and purification 87
References 89
4 ARM complexes for in vitro display and evolution of antibody
combining sites 91
Michael]. Taussig, Maria A. T. Groves, Morgit Menges, Hong Liu, and Mingyue He
1 Introduction 91
2 Ribosome display methodology 93
Outline of procedure 93
Primer design and single chain antibody (V /K) construction for ARM display 93
H
Generation of ARM complexes by coupled transcription/translation in vitro 96
Antigen selection of ARM complexes 98
Recovery and amplification of DNA from antigen-selected ARM complexes 100
Further ARM cycles and cloning 103
Analysis of clones encoding antibodies by ARM display 104
3 Examples of ARM display 105
ARM specificity 105
Selection of DB3R VH/K from libraries 106
4 Troubleshooting 107
Background 107
No DNA recovery 107
5 Summary 107
References 109
5 Human monoclonal antibodies to blood group antigens 111
Belinda M. Kumpel
\ Introduction 111
2 General equipment and reagents required 112
3 Selection of donor 113
Preparation of lymphocytes 113
4 EBV transformation of B cells 114
Preparation of EBV 115
EBV transformation of B cells and growth of B-LCL 116
5 Selection of antigen-specific B cells by resetting 117
IX