Canadian Council on Animal Care guidelines on: antibody production This document, the CCAC guidelines on: antibody production, has been developed by the ad hoc subcommittee on immunological procedures of the Canadian Council on Animal Care (CCAC) Guidelines Committee: Dr Albert Clark, Queen's University (Chair) Dr Dean Befus, University of Alberta (Canadian Society for Immunology representative) Dr Pamela O'Hashi, University of Toronto Dr Fred Hart, Aventis Pasteur Dr Michael Schunk, Aventis Pasteur Dr Andrew Fletch, McMaster University Dr Gilly Griffin, Canadian Council on Animal Care In addition, the CCAC is grateful to the many individuals, organizations and associations that pro- vided comments on earlier drafts of this guidelines' document. In particular thanks are extended to: the Canadian Society of Immunology; the Canadian Association for Laboratory Animal Science and the Canadian Association for Laboratory Animal Medicine; Dr Terry Pearson, University of Victoria; Dr Mavanur Suresh, University of Alberta; Drs Ernest Olfert and Barry Ziola, University of Saskatchewan; and Dr Patricia Shewen, University of Guelph. © Canadian Council on Animal Care, 2002 ISBN: 0-919087-37-X Canadian Council on Animal Care 315-350 Albert Street Ottawa ON CANADA K1R 1B1 http://www.ccac.ca CCAC guidelines on: antibody production, 2002 TABLE OF CONTENTS A. PREFACE . .1 E. REFERENCES . .20 B. INTRODUCTION . .2 1. General . .20 2. Polyclonal Antibody Production . .21 C. POLYCLONAL ANTIBODY PRODUCTION 3 3. Monoclonal Antibody Production . .25 3.1 Other useful references . .27 1. Animal Selection and Care . .3 3.2 Additional useful information . .27 2. Immunization Protocol . .6 3. Standard Operating Procedures . .6 F. GLOSSARY . .27 4. Immunogen Preparation . .6 5. Choice of Adjuvant . .7 6. Route of Injection . .9 7. Volume and Number of Injection APPENDIX A Sites . .10 8. Blood Collection . .12 COMMON ADJUVANTS . .31 9. Monitoring of Animals . .13 10. Disposition of the Animals . .13 APPENDIX B IMMUNIZATION – RECOMMENDED D. MONOCLONAL ANTIBODY ROUTES AND VOLUMES PRODUCTION . .14 (adapted from Leenars, et al., 1999) . .33 1. Animal Selection and Care . .16 2. Production of Hybridoma Clones . .17 APPENDIX C 3. Ascites Production . .17 STAGES OF MONOCLONAL 3.1 Priming . .17 ANTIBODY PRODUCTION . .35 3.2 Contamination . .18 3.3 Hybridoma implantation . .18 APPENDIX D 3.4 Monitoring the animals and endpoints . .19 INFORMATION ON IN VITRO 3.5 Ascites tumor growth . .19 TECHNIQUES FOR MONOCLONAL 3.6 Ascites fluid collection . .20 ANTIBODY PRODUCTION . .37 antibody production A. PREFACE on antibody production given in the 1991 CCAC policy statement on Acceptable The Canadian Council on Animal Care Immunological Procedures. (CCAC) is responsible for overseeing animal use in research, teaching and test- Many institutions already have excellent ing. In addition to the Guide to the Care and standard operating procedures (SOPs) in Use of Experimental Animals,Vol. 1, 2nd place for the production of both pAbs and Edn., 1993 and Vol. 2, 1984, which lay mAbs. These CCAC guidelines borrow down general principles for the care and much from the experience of these institu- use of animals, the CCAC also publishes tions, and from the recent international guidelines on issues of current and emerg- initiatives to refine protocols for antibody ing concerns. The CCAC guidelines on: production. antibody production is the fifth of this series, and has been developed by the CCAC ad The refinement of animal use in research, hoc subcommittee on immunological proce- teaching and testing is an ongoing process dures. which is never complete. The CCAC recog- nizes that in moving towards implementa- The purpose of this document is to present tion of these guidelines, considerable guidelines for production of both polyclonal expenditure may be involved; for example, (pAb) and monoclonal antibodies (mAb) that to establish central services for production assist investigators and research support of mAbs in vitro. As with the implementation personnel to achieve an acceptable of other CCAC guidelines, institutions must immunological result with minimal discom- recognize such expenditures as responsi- fort for the animals involved. These guide- bilities if sound humane research is to be lines are also provided to assist animal carried out within their facilities. The CCAC care committee (ACC) members to evaluate is committed to assisting institutions by protocols involving the production of providing information on production of mAbs antibodies, to ensure that the highest in vitro as well as on best practices for standards of animal care and use are met. animal-based production for pAbs and These guidelines supercede the guidance mAbs where necessary. B. INTRODUCTION and from a single animal over time. There- fore, pAbs have a finite availability and are Antibodies are produced by the immune subject to possible character change during system of an animal in a specific response the period of production. On the other hand, to a challenge by an immunogen. The monoclonal antibodies (mAbs) are derived immune system acts through two principal from a single clone and hence are specific mechanisms: humoral type responses for a single epitope and have a defined (production of antibodies) and cell-mediated affinity for that epitope. Thus if the right responses. Immunogens (antigens) are mAb is obtained, it can be extremely speci- molecules which can induce a specific fic for the relevant immunogen, and under immune response and are usually foreign appropriate conditions, an almost limitless production of a constant product is possible. ccac guidelines proteins or carbohydrates, or sometimes lipids and nucleic acids. The immune systems of mammals are comprised Polyclonal antisera can be obtained in a of large numbers of lymphocytes, each relatively short time frame (1-2 months), in characterized by its unique antigen-receptor contrast to standard mAb production specificity. This receptor diversity permits procedures that can be tedious and require immune responses to a broad range of 3-6 months. (Newer procedures, however, immunogens. The B lymphocytes, charac- can shorten the time for mAb derivation to terized by the presence of specific immuno- as little as one month.) Polyclonal anti- globulin receptors on their surface, are bodies show different affinities for different responsible for production of the humoral epitopes and thus may demonstrate overall (antibody) response. Antibodies are secret- excellent binding achieved by adherence to ed from plasma cells which have differen- a number of different sites on a complex tiated from B lymphocytes after appropriate immunogen or antigen. In contrast the stimulation by the foreign immunogen. single epitope specificity of mAbs may Each antibody molecule is capable of mean that a slight change in the structure of recognizing a specific epitope (antigenic the epitope, for example by antigen denatu- site), usually 5-6 amino acids or monosac- ration (such as occurs in immunoblotting charide units which are either linear or experiments or by sterilization of the topographically assembled, and is capable immunogen prior to injection into the of binding to this epitope. animal), can result in loss of antibody binding. For this reason two or three mAbs A polyclonal humoral response is comprised are sometimes combined. of antibodies, derived from various clonal populations having varying specificities (for Consideration should be given at the outset different epitopes, even on the same mole- to procuring commercially produced anti- cule), affinities and classes, and hence bodies from sources that comply with CCAC provides an effective defense against guidelines, or if international, are accredited pathogens. Polyclonal antisera are difficult by the Association for Assessment and to reproduce because of the variety of Accreditation of Laboratory Animal Care, antibodies made in the polyclonal response. International (AAALAC). The Antibody The level and quality of the antibodies Resource Site http://www.antibodyresource. produced will vary from animal to animal, com may assist in this regard as well as 2 provide background information on antibody immunogens or antigens (Hanly, et al., production. A list of suppliers of mAbs 1995). A number of references to suitable produced in vitro has been compiled by the immunology texts are given in the reference Focus on Alternatives Group, UK, and a list: Abbas, et al., 2001; Anderson, 1999; second list of US suppliers has been com- Kuby, 2000; Roitt, et al., 1998; Sharon, piled by the Alternatives Research and 1998; Sompayrac, 1999. Development Foundation, US. Both lists antibody production, 2002 can be found at http://www.frame.org.uk/ General guideline: In the production of Monoclonal_Suppliers.htm. Additional polyclonal antibodies, the overriding material, for example SOPs, are available consideration must be to minimize pain on the CCAC website http://www.ccac.ca. and distress for the animals used. 1. Animal Selection and Care C. POLYCLONAL ANTIBODY PRODUCTION Guideline: Careful consideration should be given to the selection of the species Polyclonal antibodies (pAbs; antisera) have to be used for polyclonal antibody a number of uses in research, for example: production. for detection of molecules in ELISA-type assays; in Western blots; in immuno- Careful consideration should be given to the histochemical and immunoprecipitation appropriateness of the species and strain procedures; and in immunofluorescence