Approved June 28, 2000.

Immunization of an animal is usually successful in generating an antibody response, the elements of which subsequently can be collected as antiserum to provide reagents used in research and/or clinical applications. These reagents are used in Western blotting, immunohistochemistry, ELISA format, and cloning protocols.

Some factors to be considered in the generation of antisera are:

• Source of antigen will determine the degree that it is perceived as foreign. This criteria helps determine the species to be considered for immunization. (Mice, rats, guinea pig, hamster, rabbit, goat, etc.) Caution should be taken to ensure that the preparation is physiologically neutral in order to avoid unnecessary tissue damage, or peritonitis.

• Forms of antigen: The more complex the molecules are the more immunogenic they will be. Purity affects the extent of specificity. Adjuvants, such as alum, Ribis, or Freunds may be appropriate to enhance a response, especially if small molecules are being considered. (Refer to the policy on adjuvants/complete Freunds).

• Route of immunization will also affect the immunogenicity. Intradermal or topical application generally elicit a delayed type hypersensitivity, where as an intravascular injection provokes development of humoral antibody. Other routes include subcutaneous, intramuscular, intranasal, intraperitoneal, intravascular, intracranial injections, as well as injection into any organ. Route/site of injection influences the class of immunoglobulin invoked in the immune response. All antigen/adjuvant injection sites should be clipped, cleaned and disinfected prior to the injection. Volume should be restricted to 0.05-0.1 ml per site distributing the preparation in numerous sites over the flank or dorsal area of the animal. Intraperitoneal volumes for rodents or hamsters should be limited to 0.5 ml. Intraperitoneal use requires detailed scientific justification approved by the IACUC in advance. (Refer to policy on adjuvants).

• Dose: A low dose of antigen may induce a delayed type hypersensitivity, but a higher dose of the same antigen may induce an antibody response. Too low or too high a dose may induce tolerance. A range of 50-75 ug /per injection for rodents, and 100ug for larger species is recommended.

• Time interval: Antibody isotype and titer change with time as the response matures. A 2-3 week interval between immunizations is suggested for reinforcing an IgG response.

• Frequency of exposure: Multiple exposures may raise antibody titer and enhance specificity, but can also stimulate control pathways to dampen a response. Immunization strategy may need to be reassessed if, after 2-3 immunizations, some degree of response is not evident.

• Monitoring of response: Testing the serum for antibody should always be done 7-10 days after boosting. (Refer to policy on blood collection.)

• Other factors to be considered that may affect the humoral response include age, drugs administered, nutrition, sex, hormones, stress, radiation, etc.

• Volume of blood collected: If quantities necessary for analytical purposes exceed those recommended in blood volume section, then a larger species should be considered. Species selected should have clinical relevance if parallels are to be inferred between subject and source reagent.

• Other considerations: As in all other procedures, sites of immunization should be monitored for ulceration or infection on a regular basis. After blood collection, hemostasis should be achieved and animals must be observed for adverse reactions at 15 minutes and two hours after the injection. Anaphylaxis is a potential in some circumstances, and care should be taken to recognize the signs early enough to initiate intervening measures.

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