Approved March 22, 2000

Complete Freund's Adjuvant (CFA) is a water in oil emulsion containing killed dried Mycobacterium butyricum which is used to enhance antigenicity and stimulate an immune response greater than the antigen alone. Although they are useful and sometimes essential for producing antibodies, adjuvants, particularly Freund's complete adjuvant, have the capability of causing inflammation. The following guidelines are intended to eliminate or reduce to a minimum animal discomfort associated with the use of adjuvants. Deviations from these standards must be scientifically justified and approved by the IACUC. The protocol must include the following information: identification of antigen, the adjuvant or solution used for injections, the volume per injection site, total volume to be injected, site of injection, route of administration, and boosting schedule.

1. Alternatives which reduce the number of animals (tissue culture, chicken eggs, etc.) or utilize non-inflammatory adjuvants (Ribi, liposomes, incomplete Freund's) must be considered. CFA should be used only when absolutely necessary, such as small amounts of available antigen and weak antigens. The use of CFA must be justified in the protocol and approved by the IACUC.

2. If CFA is used, it must be limited to the initial immunization only. Booster injections should use incomplete Freund's adjuvant or saline solution.

3. Acceptable sites for injection include the backs and flanks. All injection sites must be clipped, cleaned, and disinfected. If surgery is necessary to facilitate lymph node injection, it must be performed aseptically by appropriately trained personnel, according to guidelines for survival surgery. INJECTION OF CFA INTO THE FOOTPAD IS NOT PERMITTED because additional trauma may be produced by the animal's normal processes of weight bearing and location, limited tissue space is available for the inflammatory response, and the injection site would be exposed to the cage surface increasing the chance for infection.

4. A ratio of 1:1 of antigen : CFA emulsified mixture is standard. The maximum volume of CFA permitted per injection site is:

   Subcutaneous - 0.1 ml: Intramuscular - 0.2 ml: Intradermal - 0.05 ml

   In addition:

   • Multiple injection sites must be separated from each other widely enough to insure continued blood supply.
   • CFA must never be given intravenously.
   • If scientific evidence is provided that intraperitoneal injection is required for the immune response, the IACUC will consider intraperitoneal injections on an individual basis, only in mice and rats, at a volume of no more than 0.1 ml.

5. The injection site(s) must be observed by the investigator or his/her designate, at least three times a week for four weeks after each injection. If a lesion develops at any injection site, it must receive appropriate veterinary treatment.

 

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