Monoclonal Antibodies The Practical Approach Series Related Practical Approach Series Titles Antibody Engineering Immunodiagnostics Lymphocytes (2e) Immunoassay* Protein Localization by Fluorescence Microscopy Immunodiagnostics Immobilized Biomolecules in Analysis Complement MHC1 MHC2 Affinity Separations Immunochemistry 1 Immunochemistry 2 Antibody Engineering HIV Volume I HIV Volume II Cytokines (2e) Histocompatibility Testing * 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 UNIVERSITY PRESS OXFORD UNIVERSITY PRESS Great Clarendon Street, Oxford 0X2 6DP Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide in Oxford New York Athens Auckland Bangkok Bogota Buenos Aires Calcutta Cape Town Chennai Dar es Salaam Delhi Florence Hong Kong Istanbul Karachi Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi Paris Sao Paulo Singapore Taipei Tokyo Toronto Warsaw with associated companies in Berlin Ibadan Oxford is a registered trade mark of Oxford University Press in the UK and in certain other countries Published in the United States by Oxford University Press Inc., New York © Oxford University Press, 2000 The moral rights of the author have been asserted Database right Oxford University Press (maker) First published 2000 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, or under terms agreed with the appropriate reprographics rights organization. Enquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above You must not circulate this book in any other binding or cover and you must impose this same condition on any acquirer 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