Antibodies are important tools for research. Depending on research needs, antibodies may be produced by polyclonal or monoclonal technique. Each technique requires that specific issues be addressed in animal protocols. IACUCs should ensure adequate training of personnel in the use of proper technique when any method of immunization is proposed. The advantages of a centralized service utilizing skilled technicians to meet multiple research groups’ needs for polyclonal and monoclonal antibodies is another refinement which may enhance animal welfare in larger research programs. There are also many commercial sources of antibodies made to order.
A good resource is “Information Resources for Adjuvants and Antibody Production: Comparisons and Alternative Technologies. AWIC Resource Series, No. 3. August 1997. Call Number: aHV4701.A94no.3. ISBN 090076791X. The document includes over 500 current bibliographic cita-tions regarding adjuvants and antibody production methods compiled from scientific journals, proceedings and newsletters. A company/institute listing of suppliers of antibodies and antibody production products is included. Emphasis is placed on citing comparative studies and research into alternative methods.
Polyclonal Antibody Production
Injection of an immunogen (e.g. protein, virus, bacterium) into an animal produces a humoral response, which induces the production of a population of heterogeneous antibodies, with varying specificities toward different molecular regions (epitopes) of the immunogen. Two types of lymphocytes (T cells, derived from the thymus, and B cells, derived from marrow) are responsible for the production of polyclonal antibodies. Polyclonal antibodies produced in response to infection can be effective in recognizing and eliminating foreign material, but the heterogenicity of the product limits its use in research and industry.
To increase the immune response, the immunogen may be combined with an adjuvant. Adjuvants stimulate the rapid and sustained production of high titers of antibodies with high avidity. Adjuvants may facilitate the immune response through three basic mechanisms:
Adjuvants may serve as a depot for the antigen, which should increase the duration of antigen exposure and the antibody response.
Adjuvants may stimulate immune cells.
Adjuvants may enhance macrophage phagocytosis after binding the antigen as a particulate (a carrier/vehicle function).
The use of adjuvants is required for many antigens which by themselves are weakly immunogenic. Adjuvant selection remains largely empirical. Antigens that are easily purified or available in large quantities may be good choices for starting with the least inflammatory adjuvants for immunization. Should antibody response not be suitable, a gradual increase in the inflammatory level of the adjuvant would then be warranted.
The choice of the appropriate adjuvant is important from both the aspect of the end result (high antibody response) and the welfare of the immunized animal. Many of the adjuvants have the capacity to cause inflammation, tissue necrosis and pain in animals. A major charge to investigators is to minimize animal use and discomfort.
Freund’s incomplete adjuvant (IFA) is a water/oil emulsion containing immunogen, paraffin oil and an emulsifying agent. Addition of killed myco-bacteria to the oil phase (Freund’s complete adjuvant, CFA) enhances the immune response. Multiple exposures to CFA will cause severe hyper-sensitivity reactions. The use of CFA can be painful and alternative adju-vants should be considered. Abscesses, granulomas and tissue sloughs may occur at injection sites. However, a recent report (Halliday) suggests that when the NIH intramuralguidelines are meticulously followed, assuring aseptic technique and adding the judicious use of chemical sedation, the use of CFA for immunization is a humane procedure. Undesirable and painful side effects must be minimized or eliminated by careful preparation of inoculum, the use of appropriate routes of administration, adequate separation of injection sites, and the use of a small amount of inoculum per site.
Because of the severity of the secondary immune response to mycobacte-rium in CFA, IFA must be used with booster antigen administrations in cases where CFA has been used in the initial injection.
For many years CFA was the only effective adjuvant, but this is no longer true. Other adjuvants are available as alternatives and may be suitable for use in an investigator's experiments.
The range of recommendations for routes and sites of administration of antigen-adjuvants preparations, volumes per site and number of sites per animal for different species vary in the literature and institutional guidelines. Particularly with the use of CFA, it is important to note that the severity of potentially painful inflammatory reactions may be minimized by injection of a small volume of inoculum per site and the use of multiple injection sites when appropriate. Injection sites must be sufficiently separated to prohibit coalescing of the inflammatory lesions.
Using multiple sites for immunization also provides more foci for antigen presentation and the involvement of more lymph nodes. Intradermal and subcutaneous routes are commonly used to take advantage of antigen-processing dendritic cells present within the dermis. Hair should be clipped from intradermal and subcutaneous injection sites, and the site should be aseptically prepared with betadine or nolvasan scrub followed by alcohol or other appropriate antiseptics. The following recommendations apply primarily to antigen solutions in CFA or IFA. Volumes ranging from 0.05 ml to 0.10 ml per site have been recommended for intradermal injections in rabbits. A total of five intradermal sites has been recommended. Because intradermal sites ulcerate with FCA, sterile inocula must be used and the site must be properly disinfected to prevent secondary bacterial infection. Subcutaneous injection volumes in the rabbit vary from recommendations of 0.10 ml to 0.25 ml to 0.40 ml per site. Number of sites recommended varies from 4 to 10.
Footpad injections in rabbits are prohibited. Where scientific justification is provided, footpad injections may be permitted in rodents, but only in one hind foot, and with the animals housed on soft bedding. Suggested maxi-mum injection volumes can range from 0.01 to 0.05 for mice and 0.10 ml for rats. The need for footpad injections must be critically evaluated by the IACUC before approval.
Sometimes direct inoculation into lymph nodes, such as the popliteal lymph node, is used. With practice these nodes often can be palpated and the injection performed percutaneously.
Intramuscular injections, usually made in the biceps femoris or quadriceps muscle mass, generally are lower volumes of 0.25 ml to 0.20 –0.40 ml. Care must be exercised to avoid adjacent nerves and blood vessels as well as fascial planes when injecting into a muscle bundle. Disagreement exists as to the appropriateness of intramuscular injection of CFA. The intramuscular route of injection is recommended in some institutional guidelines and specifically discouraged in other guidelines. Intramuscular injection is generally not recommended in rodents because of limited muscle mass.
For TiterMax®, intradermal, subcutaneous, and intramuscular routes are recommended with volumes per injection site ranging from 0.01 to 0.25 ml in small and large animals. For Ribi®, intradermal, subcutaneous and intramuscular routes are recommended with volumes per injection site ranging from 0.05 to 0.50 in small and large animals.