GUIDELINES FOR POLYCLONAL ANTIBODY PRODUCTION IN LABORATORY ANIMALS Revised and approved May 2009 1. PURPOSE. Rodents and rabbits are often used to produce antibodies for a variety of research objectives. Adjuvants are used to boost the immune response. Improper or unnecessary use of Freunds complete adjuvant (CFA) may cause inflammation, induration, or necrosis. Disseminated granulomas have been reported in lungs, liver, kidney, heart, lymph nodes and skeletal muscle after subcutaneous or intravenous injection of rabbits and rats, with similar results in hamsters, mice and guinea pigs. Accidental injections in humans can result in long-term, painful abscesses. The following guidelines are intended to eliminate or reduce to a minimum, animal discomfort associated with the use of polyclonal antibody production in laboratory animals. 2. CHOICE OF SPECIES AND STRAIN. The choice of species and strain must be justified by the investigator in the Master Animal Use Protocol (MAUP). Ex vivo methods of antibody production or off-the-shelf commercially available antibodies should always be considered before in vivo antibody production. When commercial sources are used, refer to the ACUC guidelines for custom antibody production. 3. IMMUNIZING ANTIGEN. The purity and method of preparation of the immunizing antigen is extremely important. The immunizing material must be virtually free of toxic substances (e.g., urea, acetic acid). It should present no risk of pathogenicity or toxicity to the host animal, other animals in the colony, or personnel. If toxic or pathogenic immunogens must be used, they must be justified in the MAUP and approved by the ACUC. Proposed arrangements for animal housing, monitoring, and antibody procedures must be fully documented. Information regarding methods of immunogen preparation can be found in Table 4. 4. ADJUVANTS. Although Freund adjuvants are potent stimulators of the immune response their use can be problematic: it can be difficult to prepare stable emulsions with them; they are not cleared from the injection site; and they can cause ulceration, granuloma formation, untimely ascites production, and other adverse responses in laboratory animals. If Freund adjuvants are used, the “complete” adjuvant can be used only for the first (priming) immunization. Personnel using complete Freund adjuvant should be particularly careful to avoid accidental self-injection with needle tips, which causes a protracted, painful inflammation at the injection site. The proven safety and efficacy of other adjuvants such as Ribi and TiterMaxTM makes it difficult to justify use of Freund adjuvants. Because of the potential adverse effects of Freunds adjuvants, and the availability of other, potentially less harmful adjuvants, the justification for use of Freunds adjuvant must be included in the MAUP and approved by the ACUC. Information about alternative adjuvants can be found in Table 5. 5. IMMUNIZATIONS. Injections for routine antibody production should be administered subcutaneously in two to four sites per animal, generally on the back, away from the spine. Other routes such as intradermal, intramuscular, intraperitoneal may also be used, although the intradermal route should not be used in mice and intramuscular injections should not be used in small rodents. Recommended injection volumes and amounts are listed in Table 1; maximum injected volumes are listed in Table 2. The inoculum should be free of extraneous microbial contamination. Filtration (0.2 micron) of the antigen before it is mixed with adjuvant is recommended. Injection sites should be free of debris and disinfected with alcohol, Betadine or chlorhexidine. As with all guidelines, investigators who find a recommended procedure to be incompatible with the scientific needs of their research may submit a written request for the ACUC to consider an exception to these guidelines. 2 Intravenous (antigen only), intrasplenic, footpad, and popliteal lymph node injections are allowable, but generally not necessary. Use of these alternative routes must be justified in the MAUP, and approved by the ACUC. “Footpad” injections in rabbits are not acceptable due to the lack of anatomical structure. Protocols for these types of injections should clearly describe the experimental objective and details of the antigen and the entire procedure, including monitoring of the animals after injection. No adjuvant should ever be administered i.v. Table 1. Recommended immunization sites and injection volumes for injection of immunogen/depot forming adjuvant. Always use the smallest inoculum and total volume possible. Mice Rats Hamsters Guinea pigs Rabbits Subcutaneous 0.1 ml/site 4 sites max 0.1-0.2 ml/site 4 sites max 0.1 ml/site 4 sites max 0.1-0.2 ml/site 4-6 sites max 0.1-0.25 ml/site 8-10 sites max Intramuscular 0.05 ml if required: Not recommended Not recommended 0.0