Polyclonal Antibody Production
Animal Model Selection:
Rabbits are the most commonly used laboratory animal species for polyclonal antibody production due to optimum size, ease of handling, the availability of specific pathogen free rabbits and capability of producing moderate quantities of high titer and high affinity antibody from a range of antigenic material. Other species also used for polyclonal antibody production include mice, hamsters, rats, guinea pigs, goats, sheep, and chickens.
Antigen Quality and Quantity:
The antigen should be free of extraneous microbial contamination; sterilization by filtration through a 0.22 micron filter is recommended. The presence of byproducts, such as polyacrylamide gel, particulate matter and chemicals such as SDS, urea, acetic acid, or other solvents or potentially toxic agents or extremes in pH should be avoided. Special precautions may be necessary if the antigen itself is a viable microbe. Generally the quantity of protein providing the optimum dose in rabbits range from 50 to 1000 ul., and the lower range may result in higher affinity antibody production.
Freund's Adjuvant and Alternatives:
Freund's Complete Adjuvant (FCA) is commonly used for polyclonal antibody production. Due to the inflammatory reactions associated with FCA, many sequelae have been described which include granulomatous inflammation, focal necrosis, ulceration of skin, fistulous tracts, muscle atrophy, self-induced trauma, hypersensitivity reactions, and weight loss. For these reasons, alternative adjuvants with less inflammatory potential than FCA, should be used, if suitable. Ribi Adjuvant System® and TiterMax® are commonly cited as appropriate alternatives. Non-inflammatory adsorptive adjuvants such as alum and aluminum hydroxide gel may also be considered. FCA is specifically discouraged for use in nonhuman primates because of subsequent interference with routine tuberculin skin tests used for diagnostic purposes.
Inoculation Methods:
FCA is used only once per animal, usually for the initial immunization. Freund's Incomplete Adjuvant (FIA), which contains no mycobacterial cell mass, is commonly recommended as the adjuvant for booster immunizations following FCA to preclude hypersensitivity reactions. For Freund's adjuvant immunization protocols, the recommended interval between primary inoculation and booster immunizations range from 3-6 weeks or longer. It is recommended that booster immunizations be delayed if significant inflammatory reactions are still present from the previous immunization. Also for adjuvant-antigen mixtures that cause excessive inflammatory reactions, consideration should be given to using lesser concentrations, smaller volumes per injection site, or both for subsequent immunizations.
Most common inoculation routes for non-rodent species include intradermal, subcutaneous and intramuscular. Particularly with the use of FCA, it is important to note that the severity of potentially painful inflammatory reactions may be minimized by administering small volumes per site and use of multiple sites. The intradermal route may be the most favorable for initial priming doses because of efficient lymphatic delivery. In the rabbit, recommend 0.05 - 0.1 ml and 5-10 sites. It is important to adequately separate injection sites to preclude coalescing of inflammatory lesions which may result in disruption of blood supply with subsequent formation of abscesses and tissue sloughs. Injections by the s.c. route are convenient and will accommodate larger volumes but absorption is generally slower. Acceptable range of volume/site in rabbits is 0.1-0.25 ml per site; 4-10 sites distributed over the four corners of the inguinal and auxillary regions. Injections by the i.m. route for FCA are suggested by some as a site that provides rapid absorption, but it also produces severe muscle necrosis, especially with larger volumes >0.25 ml/site, and, thus, this route is not routinely recommended for FCA. When i.m. inoculations are administered, 0.1-0.25 ml/site is recommended and avoid the paraspinal muscles. The i.m. and perhaps the s.c. routes may be more appropriate for booster inoculations with FIA because distribution from i.d. sites may be more localized with subsequent injections. Anatomic sites used for grasping, handling, or restraint of animals such as the dorsal cervical/scapular areas and rump in rabbits and dorsal cervical/scapular areas and tail base in rodents should be avoided as inoculation sites when possible. "Footpad" injections/immunizations in rabbits are unsuitable because of the lack of anatomically defined footpads, and the weight bearing function of their feet. Subcutaneous and intraperitoneal routes are more common for rodents. Suggested maximum injection volumes can range from 0.01 to 0.05 ml for mice and 0.10 ml for rats. Intramuscular routes are avoided in rodent due to limited muscle mass. Where scientific justification is provided and approved by the IACUC, footpad injections may be permitted in rodents, but only in one hind foot, and with the animals housed on soft bedding. Suggested sites vary depending on whether adjuvants are used and if so what type and the quantity and type of antigen available. Less common sites include intrasplenic or intranodal, when limited quantities of antigen are available. The intravenous (i.v.) route may be acceptable for wide distribution of soluble antigens. However the i.v. route is NOT appropriate if the antigen is combined with an adjuvant.
Specific administration methods - intradermal & subcutaneous sites. Clip hair and aseptically prep sites to reduce the potential for the development of infection and abscess formation. Clipping also facilitates visualization of the injection sites to permit appropriate treatment of lesions if they develop. Use sterile needles (25 gauge for i.d. & 20-23 gauge for s.c. routes) and syringes to minimize microbial contamination of injected tissue. Intramuscular sites: Prep the injection site with an antiseptic (i.e. alcohol). Injections are usually made in the biceps femoris or quadriceps muscle mass. Use sterile needles (20-23 gauge) and syringes.
For adjuvants other than Freund's adjuvants, such as Ribi Adjuvant System® and TiterMax®, the manufacturer provides detailed information on preparation of antigen-adjuvant emulsions, recommended routes and sites, total volume and volume per site for injection in different species, and recommended immunization schedules.
Post-injection Observations:
Investigators and veterinary staff should observe animals for evidence of pain or distress, and for evidence of lesions such as swelling, abscess or fistula formation, and infection or ulceration at the immunization sites. Follow-up should include clinical observations and palpations of the injected sites with emphasis on determining the necessity for any supportive therapy. Investigators should contact the veterinary staff if injection site lesions or evidence of pain or distress are identified in any animals. This will permit timely and appropriate assessment and institution of therapy when required. Supportive therapy may include topical cleansing, antibiotic administration and/or analgesic administration.
Blood Collection:
Refer to the IACUC statement on approved blood collection techniques including methods and routes of collection for both survival and terminal procedures.
