Date: Revised July 2009
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 APLAC 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 (725-9901).

An important aspect of adjuvant immunization is the utilization of competent and skilled technical staff with experience handling the species being used and in performing the technique. They must be knowledgeable and capable of recognizing signs of distress in all injected animals, and be responsible for taking action when necessary. Improper or unnecessary use of Complete Freund's Adjuvant (CFA) may cause inflammation, induration, or necrosis in laboratory animals. Disseminated granulomas have been reported in lungs, liver, kidney, heart, lymph nodes, and skeletal muscle after subcutaneous or intravenous injection in rabbits and rats, with similar results in hamsters, mice, and guinea pigs. [1] Humans accidentally injected have suffered long-term, painful abscesses [2] , therefore personnel should use care when preparing and injecting adjuvants into animals. The APLAC has developed the following guidelines intended to eliminate, or reduce to a minimum, animal discomfort associated with the use of this agent in research.

1. Before using CFA, consider the use of Incomplete Freund’s Adjuvant (IFA) or other alternative and effective adjuvants. Use of TiterMaxTM from CytRx®, RIBI Adjuvant System, AlhydrogelTM, LipovantTM, AdjuvaxTM, or other commercially available immunoadjuvants may be acceptable alternatives [3, 4]. However, some studies have shown CFA:IFA to be the superior adjuvant [4]. If using another acceptable adjuvant, follow the 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 CFA 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, with IFA the adjuvant of choice for subsequent immunizations. Use of two or more doses of CFA must be justified, in writing, described in protocol, 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 CFA, and suggested blood withdrawal maximums, are on the table below. If the Protocol Director (PD) would like to use a combination of differing routes of injection, please contact the VSC (723-3876) for assistance.
   • Use of footpad injections is not recommended and will not be allowed unless scientific proof is provided, in writing, described in protocol, 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 the VSC for ameliorative measures to be taken in the event of discomfort. Alternatively, “tail-base” injections can be used for situations where “foot pad” injections have traditionally been used (i.e., adjuvant-induced model of arthritis). Intramuscular injections of CFA are not recommended for small laboratory rodents. However, for studies tracing the fate of injected antigens, it is suggested that injections be made intramuscularly, but in areas drained by particular lymph nodes such as the axillary or inguinal.
   • The injection site must be observed by the PD or 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 (723-3876).
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. Contamination is likely to result in infection, but the injection sites 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.

(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.)