Date: October 1998
Reference:  Administrative Panel on Laboratory Animal Care

DIRECTIONS: Review following material. Keep copies of guidelines with applicable protocols. You may find it helpful to post a copy of these guidelines in your laboratory. Questions should be forwarded to the A-PLAC office, 723-4550.

TRAINING: Training in these techniques and the humane treatment of laboratory animals during the procedures is taught by the Veterinary Service Center (VSC) Staff. All new personnel who will be performing these techniques should contact VSC staff for training (723-6735).

1. Before using CFA, consider the use of incomplete FA or another adjuvant (3, 4). Use of the TiterMaxTM from CytRx®, RIBI Adjuvant System or other commercially available immunoadjuvants may be acceptable alternatives. However, some studies have shown CFA:IFA to be the superior adjuvant (4). If using another acceptable adjuvant, follow manufacturer's instructions for its use. Please include a description of the antibody production regime in your protocol under "Procedures."
   
   
2. Undesirable and painful side effects of complete Freund's adjuvant can be effectively reduced or eliminated by the use of appropriate routes of administration, adequate separation of injection sites, and the use of small amounts of inoculum per site. If CFA must be used:
• It should be used only for the first (priming) antigenic dose. Use of two or more doses of the complete adjuvant must be justified, in writing, (attach to these guidelines) and is rarely warranted. If more than one dose must be used, an interval of at least three weeks should be allowed between doses.
• Suggested maximum immunization volumes for mice, rats, and rabbits, using Complete Freund's adjuvant, and suggested blood withdrawal maximums are on the reverse side of this form. If the Director wouldlike to use a combination of differing routes of injection, please contact VSC, 723-6735, for assistance.
• Use of footpad injections is not recommended and will not be allowed unless scientific proof is provided, in writing (attach to these guidelines), indicating that this route is specifically required for sufficient antibody production. Injection of a rear footpad in mice or rats may be permitted if other sites do not produce significant antibody titers to weak antigens. If footpads must be used, contact VSC for ameliorative measures to be taken in the event of discomfort. For studies tracing the fate of injected antigens, it is suggested that injections be made intramuscularly in areas drained by particular lymph nodes such as the axillary or inguinal.
• The injection site must be observed by Director his/her designate a minimum of three times per week for four weeks after each injection. Written documentation of these observations should be maintained. Any abnormalities noted during the observations must be reported to the VSC staff (723-6735).


3. The inoculum should be free of extraneous microbial contamination. Millipore filtration of the antigen before mixing with adjuvant is recommended when possible.
   
   
4. Injection sites should be clean and free of debris and contamination likely to result in infection, but need not be aseptically prepared.



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References


1. Schiefer, B. and Stunzi, H.: Pulmonary Lesions in Guinea Pigs and Rats After Subcutaneous Injection of Complete Freund's Adjuvant or Homologous Pulmonary Tissue. Zbl. Vet. Med. A. 26:1-10, 1979.
   
   
2. Chapel, H.M. and August, P.J.: Report of Nine Cases of Accidental Injury to Man with Freund's Complete Adjuvant. Clin. Exp. Immunol. 34:358-541, 1976.
   
   
3. Leenaars, P.P., Hendriksen, C.F., Angulo, A.F., Koedam, M.A., Claassen, E.: Evaluation of Several Adjuvants as Alternatives to the Use of Freund's Adjuvant in Rabbits. Veterinary Immunology and Immunopathology, 40(3):225-41, 1994.
   
   
4. Johnston, B.A., Eisen, H., Fry, D.: An Evaluation of Several Adjuvant Emulsion Regimens for the Production of Polyclonal Antisera in Rabbits, Laboratory Animal Science, 41(1):15-21, 1991.
   




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Suggested Maximums for Immunization Protocols using Freund's Adjuvant
(Note: Freund's can only be used for the priming dose)


(For use of other adjuvants, follow manufacturer's instructions and submit antibody production regime under point 3. "Procedures" of protocol form.)