Office of Research Integrity

Acceptable Immunological Practices

*MUSC has adopted the Canadian Council on Animal Care Guidelines on Acceptable Immunological Procedures. DLAR encourages investigators to try adjuvants other than Freund's. The Ribi Adjuvant System a and Hunter's Titer Max b are synthetic adjuvants associated with fewer adverse effects than FCA.


There are no recommendations limiting the number of booster immunizations. The interval between boosts should be based upon the time required for the animal to process the immunogen and mount a response. Animals with lesions resulting from previous immunizations must not be boosted by the same route prior to resolution of pre-existing lesions.

When beginning an immunization, choosing the correct adjuvant may be difficult. As a general suggestion, Freund's Complete Adjuvant (FCA) may be used when only small amounts of soluble immunogens are available. FCA is considered to be an emulsion consisting of equal volumes of FCA to antigen (1 part FCA or less to 1 part antigen). If large amounts of particulate, or highly immunogenic immunogens are available, other adjuvants should be considered.

An important aspect in immunization procedures is the utilization of skilled, competent, technical staff experienced in the handling of 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.

FCA should be used only for the most problematic immunization situations. It must never be given either intravenously or in repeated doses. FCA must not be used in horses.

Intradermal Route:

Sound scientific evidence and justification must be available if the intradermal route of injection of FCA is to be used, because of the frequent ulceration and infections that occur at the site of such injections. The use of the intradermal route may be justified only when the purpose is to induce cell-mediated response.

In rabbits, volumes of inoculum in excess of 0.05 mls (50 microliters) per site should not be used. The location of the site(s) should be carefully selected so as to prevent mutilation. A minimal number of sites should be selected, and the distance between each site be maximized.

The intradermal route is inappropriate in the mouse. Nor is it recommended in other rodents.

Subcutaneous Route:

In guinea pigs, up to a total volume of 0.4 ml (400 microliters) of inoculum may be injected subcutaneously dorsally in the neck, in one or divided into several sites. In rabbits, the site of choice is the interscapular region (between the shoulder blades) on the dorsum (back), administering up to 0.25 ml of inoculum (250 microliters) per site, to a maximum of four sites. The distance between sites should be maximized. In the mouse, up to 0.1 ml (100 microliters) may be administered in the neck region.

Intramuscular Route:

In rabbits, intramuscular injections of FCA may be administered in the thigh muscle; up to .5 ml (500 microliters), preferably in one site. Intramuscular injection of FCA is not recommended for small laboratory animals such as rats, mice, hamsters, gerbils, etc. For larger animals, such as cats, dogs and poultry, up to 1 ml of FCA injected into the thigh muscle is acceptable. In livestock such as pigs, cattle, sheep and goats, the intramuscular route is acceptable.


The intraperitoneal route for injection of FCA is permitted on small rodents only. FCA should be administered only once and be limited to minimal volumes of up to 0.1 ml (100 microliters).

Intravenous Route:

Freund's Complete Adjuvant is not to be injected intravenously.

Footpad Injection:

Footpad injection is not permissible in the rabbit

Footpad injection of CFA in rodents is not permissible unless there is scientific proof indicating this route is essential as a specific requirement for the production of antibodies. In rats and mice, only one footpad may be used. Animals should be maintained on soft bedding and not on wire-bottomed cages. Subcutaneous injection near the footpad in the lateral tarsal region are considered to be as effective as footpad injections and are allowed.

In avian species, the common routes of injection of FCA are intramuscular and subcutaneous.

Observation of Injection Sites:

The injection site(s) must be observed by the investigator or his/her designate, a minimum of three times per week, for four weeks after each injection.

If any lesion(s) develop at any injection site, it must be reported through established channels, (e.g., the animal resources supervisor or veterinarian) and must receive appropriate veterinary treatment. Such lesions should be inspected at least three times per week by the investigator or his/her designate, until all lesions are healed.


a Ribi Adjuvant System, Ribi Immunochem Research, Ind., P.O. Box 1409, Hamilton, MO 59840

b CytRx Corp., 150 Technology Parkway, Technology Park/Atlanta, Norcross, GA 30092