Florida State University
Polyclonal Antibody Production Protocol - Rabbits
While many species may be used for polyclonal antibody production (chickens, goats, guinea pigs, mice, sheep, rats, rabbits, horses and hamsters), the rabbit is the most commonly used for reasons of historical antecedents, cost-benefit ratio and ease of handling. The selection
species should be based upon several factors:
Amount of antibody required. Larger species (sheep, horses,
when large quantities of antibody are desired.
goats) are often chosen a compromise between
farm animals and rodents or chickens. Phylogenetic relationship between antigen donor and antigen producer.
the more distant the phylogenetic relationship the more potential for a high antibody
titer. Required characteristics of the antibody.
Such include the class, isotype and
complement fixing nature of the antibodies to be produced. The intended use of the antibody. For example the antigen-binding antibody may need to be from a different species than the secondary antibody in an ELISA assay. Selection of strain within a species may be important with regard to differences in the MHC complex or immune regulatory mechanisms.
Young rabbits (2.5-3.0 kg; 10-16 weeks of age) should be used. At this age maternal IgG antibodies have declined to undetectable levels while the individual’s immune system is approaching near adult levels. Older rabbits are not as useful for antibody production as immune function peaks at puberty and the ability to respond to new antigens declines with age. Female rabbits are more often used due to their docility. There are also reports that females (of many species) are sensitive to lower doses of antigen and may have significantly higher and more prolonged responses to immunization than males.
When calculating the number of animals to request, 2-3 animals per antigen is recommended to account for response failure, based upon previous reports. This is because there may be variability of the antigenic response in different individuals. Using more than one animal allows a more diverse response in terms of quantity of antibodies, specificity, and affinity. To comply with the 3Rs (reduction, replacement, refinement), if possible a sequential immunization approach should be used wherein if the first rabbit produces a high antibody titer then there is no need for a second or third animal.
Antigens used should be clean of contaminants and avoid extremes of pH. Extraneous microbial contamination, protein contaminants, chromatographic by-products (such as polyacrylamide gel) or chemical contamination (i.e., SDS, urea, acetic acid and solvents) may lead to a low titer of the desired antibody. In addition, contaminants and pH extremes can contribute to inflammatory reactions detrimental to the healthsof the animal. Sterilization by filtration through a low binding 0.22 micron filter (i.e., cellulose acetate) should be done whenever possible. Antigen prepared by gel electrophoresis should be either (1) eluted, lyophilized, ground to a fine powder, and re-suspended in sterile saline or (2) transferred to nitrocellulose paper, trimmed and cut into fine pieces.