IACUC Guideline VI
Use of Freund's Adjuvant in Animals

Background:

Freund's adjuvant, in complete (with addition of killed mycobacterium) or incomplete forms, is used to increase the magnitude and duration of the immune response to injected antigen. The primary disadvantage is that the adjuvant induces aggressive and persistent granulomatous lesions when used intradermally or subcutaneously1. In addition, intramuscular injection may cause permanent lameness, and intravenous injection may damage the lungs due to lipid embolism. Intraperitoneal injection, used in mice and rats, causes granulomatous peritonitis, but does not appear to cause chronic distress or impairment2. Because of its detrimental effects, other available adjuvant systems should be considered before using Freund's adjuvant1,3,4,5. For protocols that require the use of Freund's adjuvants, the following guidelines are provided to minimize animal discomfort. Any exceptions to these guidelines must be justified, and will require more rigorous review by the Institutional Animal Care and Use Committee (IACUC).

General:

1. To prevent infection, the inoculation site should be clean and disinfected, appropriate agents.
2. The antigen preparation to be inoculated should be sterile.
3. Appropriate housing and follow-up care should be provided to animals that have received adjuvant injections.
4. Complete Freund's adjuvant (CFA) should be used only for the initial inoculation. Subsequent booster inoculations should be done either with incomplete adjuvant or with no adjuvant.
5. Serum antibody titers should be tested at frequent intervals during the immunization regimen to ensure that the animal is subjected to the minimum number of injections needed to produce the desired state of immunization.
6. The Pain Category is E; therefore, a literature search for alternatives will be necessary.

Subcutaneous Inoculations:

1. All inoculations using Freund's adjuvant should be by the subcutaneous route, except for the use of the intraperitoneal route in mice and rats.
2. Subcutaneous inoculations should be done in areas not over bony protuberances, such as the spinal column. No injections should be done in the foot or footpad.
3. Subcutaneous inoculations in Freund's adjuvant should be divided so that no more than 0.1 ml per is injected per site, except in mice, where the volume injected per site should be limited to 0.05 ml.
4. Subsequent booster inoculations should be spaced at intervals of at least two weeks.

Intraperitoneal Inoculations (Mice and Rats):

1. Total inoculation volume should be limited to 0.2 ml or less.
2. Injections should be done with caution to avoid puncturing the intestines.
3. Subsequent booster inoculations should be spaced at intervals of at least two weeks.

References:

1. Harlow, E. and Lane, D. (1988). Immunizations. In Antibodies, A Laboratory Manual. Cold Springs Harbor Laboratory Press. 53-137.
2. Toth, L.A., Dunlap, A.W., Olson, G.A., and Hessler, J.R. (1989). An evaluation of distress following intraperitoneal immunization with Freund's adjuvant in mice. Lab. Animal Sci. 39: 122-26.
3. Cooper, H.M. and Paterson, Y. (1994). Production of Antibodies. In Current Protocols In Immunology. John Wiley and Sons, Inc. Section 2.4.
4. Lipman, N.S., Trudel, L.J., Murphy, J.C., and Sahali, Y. (1992). Comparison of immune response potentiation and in vivo inflammatory effects of Freund's and RIBI adjuvants in mice. Lab. Animal Sci. 42: 193-97.
5. Leenars, M., Koedam, M.A., Hendriksen, C.F.M., and Claassen, E. (1998). Immune responses and side effects of five different oil-based adjuvants in mice. Vet. Immunol. Immunopath. 61: 291-304.

Last Revised by the IACUC on 02/11/2002
Last Approved by the IACUC on 12/08/2008