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Policy: Freund's Adjuvant in Animals (IACUC)

Policy: Freund's Adjuvant in Animals (IACUC)


The use of adjuvants in research animals must be reviewed and approved by the IACUC. The selection of the appropriate adjuvant should be based on both the experimental purpose, such as antibody production or vaccine development, and the requirement to minimize pain and distress often associated with adjuvant use. The use of any adjuvant (e.g. Complete Freund’s Adjuvant) that has the potential to induce a severe local or systemic reaction may be permitted when adequate scientific justification is provided in an IACUC protocol. This policy provides guidelines for the use of adjuvants and specifically addresses the use of Complete Freunds’s Adjuvant in antibody production.  This policy also provides guidance for adjuvant use that will ensure animal health and welfare.


Adjuvants are utilized in vivo for the production of polyclonal antibodies to specific antigens. The in vivo immune response (cellular and/or humoral response) is enhanced by the use of adjuvants. Currently, there are many commercially available immunologic adjuvants. The choice of the adjuvant should be based on the goal of the antibody production and the level of immune response required or the purpose of the antibody production (vaccine development, autoimmune disease induction, or antibody production). It is vital to balance the need for a robust immune response with minimizing animal welfare concerns.


Adjuvant: substances that are injected along with an antigen that are intended to enhance the humoral and/or cell-mediated immune response to the antigen.

Antigen: any substance (such as an immunogen or a hapten) that evokes an immune response either alone or after forming a complex with a larger molecule.

Complete Freund’s Adjuvant (CFA): an adjuvant containing a light mineral oil, mannide monooleate (a surfactant agent), and heat killed and dried mycobacterium

Incomplete Freund’s Adjuvant (IFA): similar to CFA but lacks the heat killed mycobacterium.


Choice of adjuvants:

Complete Freund’s Adjuvant is considered to be the most potent and effective adjuvant which may be used with a wide range of antigens. While CFA is capable of stimulating high titers of antibodies, if used improperly it can lead to adverse clinical symptoms in research animals. The intense inflammatory reaction that is essential to CFA’s immune response is also the source of significant side effects.  CFA may result in a localized inflammatory and granulomatous reaction at the injection site. Additionally, chronic inflammation, skin ulceration or abscess, diffuse systemic granulomas secondary to migration of the oil emulsion, adjuvant related arthritis, and chronic wasting disease have all been associated with the use of CFA.  Also of concern with CFA use, is the possibility of human exposure due to accidental injection of CFA which may result in chronic, painful granulomas. 

In view of the fact that CFA has several potential side effects, it is important to consider other alternative adjuvants that are less inflammatory. Some alternatives to CFA include other emulsions (TiterMax, TiterMax Gold, Ribi Adjuvant System, Syntex Adjuvant Formulation (SAF), Specol, etc.), aluminum compounds (e.g., alum), monophosphoryl lipid A, etc. Recommendations regarding these alternatives are available from Division of Animal Resources (DAR) and Agricultural Animal Care and Use Program (AACUP.       

IACUC Protocol requirements when using CFA:
The use of CFA must be scientifically justified in an IACUC protocol and a search for alternatives for its use must be documented.  CFA is also considered a chemical hazard therefore, safety precautions must be addressed in the protocol. CFA should only be used for the first (priming) antigenic dose. Freund’s incomplete adjuvant (IFA) may be used for subsequent immunizations. The following information describing CFA use should be included in the appropriate protocol sections.

  • Route of administration
  • Number of injections per immunization time point
  • Location of injection sites
  • Volume to be administered
  • Concentration of CFA
    • Concentrations of <0.1 mg/ml are recommended in order to minimize the inflammation and possibility of focal necrosis.
  • Immunization frequency
    • The minimum interval for injection of CFA and IFA is 2 weeks.
  • Preparation of antigen-adjuvant emulsion
  • Preparation of injection site
  • Post-injection monitoring (clinical elements, frequency and duration of monitoring)
  • Pain relief
    • Use of analgesics and supportive care (e.g. moistened food) should be considered if pain and distress are observed

Preparation of adjuvants and antigens:

  • Preparation of all adjuvant-antigen emulsions must be performed using sterile technique.
  • Antigen ideally should be pH neutral and free from toxic solvents.
  • A 0.22 um filter should be used to reduce any possible microbial or protein contamination in the antigen.

Administration of adjuvant-antigen emulsifications

  • Sedation is strongly recommended if multiple sites are injected. 
  • The injection site should be shaved for better visualization of site post-injection.
  • Appropriate volumes and routes of administration should be used that allow for good results while minimizing pain and distress, particularly when using CFA (Table 1).    
  • The smallest gauge needle that allows delivery of the emulsification should always be used.  
  • Injection sites should be adequately separated to avoid coalescing of lesions.
  • A maximum of four subcutaneous injection sites per animals may be used.
  • Sites of injection should be chosen that do not interfere with normal locomotion or handling.

Post-injection monitoring

  • Injection sites should be closely monitored for lesion development.
  • DAR or AACUP veterinary staff must be notified promptly if lesion development occurs or if the animal exhibits signs related to pain or distress

Table 1. Recommended Volume (mls) of CFA emulsion per site and route of administration.  All routes require scientific justification in an IACUC protocol.

Route of Administration   Species       
  Mouse Rat Rabbit
Subcutaneous (SC) <0.1 ml* <0.1 ml <0.25 ml*
Intradermal (ID) Not recommended <0.05 ml <0.05 ml
Intramuscular (IM)* <0.05 ml <0.05 ml <0.25 ml
Intraperitoneal (IP) <0.2 ml <0.5 ml Not recommended

NOTE: Intravenous administration is not allowed.

Routes of Administration Presenting Special Issues

Footpad injections:
Footpad injections must not be used for routine immunization in rodents unless robust scientific justification is provided. There is increased probability that pain and impaired mobility will occur even when a single footpad is injected.  The use of hock injections site may be substituted for footpad injections when research requires the study of local immune responses in draining lymph nodes. The hock immunization technique involves injection of antigen-adjuvant into the lateral tarsal region, just above the hock and allows for harvest of the local popliteal lymph node. In one study, hock immunizations were found to be comparable to foot pad injections in terms of systemic antibody levels and local CD4+ T cell responses but with fewer welfare concerns. 

Intraperitoneal (IP) injections:
The use of IP injections of adjuvant-antigen emulsions, in order to obtain peritoneal exudate for immunological research, may only be conducted in rodents. Intraperitoneal injections may cause adhesions, granulomatous inflammation and painful abdominal distention from ascetic fluid. A description of monitoring procedures and humane endpoints must be clearly defined in an IACUC protocol. Rodents must be evaluated at a minimum of every other day during the first 5 days post-inoculation. Thereafter, daily monitoring of rodents is required. Monitoring for pain and distress must include assessment of ascites volume through determination of weight and/or ascites volume, clinical signs (e.g. body condition, respiratory difficulty, inappetence). Refer to the UIUC IACUC policy on hybridomas in rodents and the collection of ascites fluid. 


Broderson, J. R. 1989. A Retrospective Review of Lesions Associated with the use of Freund’s Adjuvant. Lab Animal Sci 39: 400-405.

Jackson L.R., Fox J.G. Institutional policies and guidelines on adjuvants and antibody production. ILAR Journal 37: 141-152, 1995.

Jennings VJ. Review of selected adjuvants used in antibody production. ILAR Journal 37: 119-125, 1995.

Kamala, T. 2007. Hock Immunization: A humane alternative to mouse footpad injections. J. Immunol. Methods 328: 204-214.

Kolstad, A.M. et al. 2012. Effect of pain management on immunization efficacy in mice. J Am Assoc Lab Anim Sci. 2012 Jul; 51(4):448-57.

NIH OACU Animal Research Advisory Committee’s Guidelines for the Use of Adjuvants in Research. 

Oscherwitz, J., Hankenson F.C., and KB Cease. 2006. Low-dose intraperitoneal Freund’s adjuvant: toxicity and immunogenicity in mice using an immunogen targeting amyloid-beta peptide. Vaccine (15): 3018-25.

Stills, H. F., and M. Q. Bailey. 1991. The use of Freund’s Complete Adjuvant. Lab Animal Sci 20(4): 25-31.

Stills H.F. 2005 Adjuvants and antibody production: dispelling the myths associated with Freund’s complete and other adjuvants. ILAR journal. 46(3): 280-293.

Toth, L. A., A. W. Dunlap, G. A. Olson, and J. R. Hessler. 1989. An Evaluation of Distress Following Intraperitoneal Immunization with Freund’s Adjuvant in Mice. Lab Animal Sci 39(2): 122-126

Approved: 11/3/2009
Updated: 4/2/2019



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