UCLA investigators are encouraged to use the information outlined below in designing the most efficient immunization protocols in terms of the efficacy of antibody production and minimizing potential pain and distress to the animals used.
Investigators are asked to consider using commercial vendors as a source of polyclonal antibodies. The cost of using commercial vendors is frequently less than generating these antibodies at UCLA. Additional information regarding off-campus vendors is available from the Division of Laboratory Animal Medicine (DLAM). DLAM also assists many investigators with technical and record-keeping procedures associated with on-campus antibody production.
Investigators are reminded that, although many adjuvants are non-pharmaceutical grade agents, the ARC requires that pharmaceutical-grade components be used when available (e.g., saline) and that methods to preclude bacterial contamination be employed (e.g., antigen filtration prior to mixing with the adjuvant). Some pharmaceutical-grade adjuvants, such as Montanide, are available and should therefore be a primary consideration when selecting an adjuvant. Although most investigators are familiar with the Freund.s adjuvants, Ribi Adjuvant System®, and Hunter's TiterMax®, investigators should familiarize themselves with other alternatives such as those briefly described below. Since the commonly used Fruend's adjuvants can result in significant pain or stress, investigators are expected to consider alternatives.
Selected Adjuvants and Their Properties
Freund's Mineral Oil Adjuvant Emulsions
Freund's complete adjuvant (FCA) is a mixture of a non-metabolizable oil (mineral oil), a surfactant (mannide monooleate), and killed mycobacteria (Mycobacteria tuberculosis or M. butyricum). Freund's incomplete adjuvant (FIA) is the same as FCA except that it does not contain killed mycobacteria. Freund's adjuvants are prepared as water-in-oil emulsions by combining approximately equal volumes of adjuvant and aqueous antigen solution in such a way that the oil becomes the continuous phase. If properly mixed, the antigen will be distributed over a large surface area, which increases the potential for interaction with relevant antigen presenting cells in vivo. Moreover, like other water-in-oil adjuvants, FCA and FIA exert an antigenic depot effect and therefore sequester and release the antigen over long periods of time, thus resulting in a prolonged antibody response. Both formulations can result in adverse reactions resulting from the stimulation of cell-mediated immune responses. FCA is particularly toxic when injected into laboratory animals because the host response to the non-metabolizable oil and the mycobacteria can result in both local and disseminated granulomatous reactions. Less severe inflammatory reactions result when: a) the concentration of mycobacteria in FCA is less than 0.5 mg/ml; b) more concentrated aqueous antigen solutions are added, resulting in an antigen-rich emulsion and reduction in the quantity of emulsion injected; c) multiple injection site s with minimal volumes of emulsion are injected at any one site; and d) the injection sites are separated to avoid fusion of inflammatory lesions. In addition, FCA is to be used only for weakly immunogenic antigens and only for initial immunizations. If booster immunizations are necessary, FIA must be used instead of FCA. The recommended volumes per subcutaneous injection site of Freund's adjuvants (FCA/FIA) are as follows:
|Species||Injection Volume (ml)||Max. injection sites|
Ribi Adjuvant Systems (RAS)
The manufacturer offers different mixtures of oil, detergent, and immunostimulator(s) from which to choose. Three RAS formulations, each containing different detoxified bacterial products as immunostimulators, are commercially available: a) mycobacterial trehalose dimycolate (TDM) emulsion recommended for use with strong immunogens in mice, guinea pigs, and rats; b) Gram-negative bacterial monophosphoryl lipid A (MPL®) + TDM emulsion recommended for use in mice, guinea pigs, and rats; and c) MPL® + TDM + mycobacterial cell wall skeleton (CWS) emulsion recommended for use in rabbits and large animals (e.g., goats). Adding aqueous antigen to the adjuvant and vortexing the mixture forms stable oil-in-water emulsions. In general, Ribi's adjuvant emulsions, like other oil-in-water emulsions, are more suitable for hydrophobic or amphipathic protein antigens than for hydrophilic proteins since the effectiveness of RAS is dependent on antigen absorption to the oil droplets in the oil-in-water emulsion. The inflammatory response associated with adjuvant use is minimized by utilizing metabolizable oil (squalene) and detoxified bacterial components as immunostimulators. Furthermore, since significantly less oil is needed to form stable oil-in-water emulsions (e.g., RAS) than for stable water-in-oil emulsions (e.g., Freund's-type adjuvants), Ribi's adjuvant causes less tissue damage when injected into animals. It should be noted, however, that since RAS has less of an antigenic depot effect than water-in-oil emulsions, more frequent booster injections are usually needed for an adequate antibody response. Immunization protocols recommended by the RAS manufacturer are:
MPL® +TDM Emulsion, R-700 or TDM, R-760*
Mouse: 0.2 ml dose, 0.1 ml administered subcutaneously in two sites
Guinea Pig, Rat: 0.4 ml dose, 0.2 ml administered subcutaneously in two sites
*Use with strong immunogens
Inject on day 0, boost on day 21, and test bleed on day 26 or 28. If necessary, booster injections may be given every 21 days using the same formulation. For monoclonal antibodies, boost intravenously (or intraperitoneally) with saline/antigen only 14 days after last adjuvant/antigen injection. Remove spleen for fusion three days following intravenous injection.
MPL® + TDM + CWS Emulsion, R-730
Rabbit: 1.0 ml dose, 0.05 ml administered intradermally in six sites, 0.3 ml administered intramuscularly into each hind leg, and 0.1 ml administered subcutaneously in the neck region.
Inject on day 0, boost on day 28, and test bleed on day 38 and 42. If necessary, booster injections may be given every 28 days using the same formulation. Note: Injecting more frequently than every 28 days may result in a reduction in the immune response.
TiterMax® is a water-in-oil emulsion, similar to Freund's adjuvants, containing metabolizable oil (squalene) and a non-ionic surfactant (copolymer of polyoxyethylene and polyoxypropylene). Most adjuvant activity is attributed to the surfactant.s ability to activate and bind certain complement components, which putatively target the antigen to follicular dendritic cells in the spleen and lymph nodes.
Some studies suggest that TiterMax® is superior to Freund's adjuvants with some protein antigens, particularly in rabbits and mice. Compared with FCA, TiterMax® can be used in smaller quantities for initial injection, which minimizes the inflammatory reaction at the injection site(s), and less frequent booster injections are required. Recommended routes of administration and injection:
|Species||Injection Route||Total Injections||Volume per Injection|
|Guinea Pig||IM||2||0.05 ml|
Montanide ISA Adjuvants®
A group of oil/surfactant-based adjuvants where surfactants are combined with a non-metabolizable and/or metabolizable oil. Components undergo quality control to protect against contaminants yielding excessive inflammation. Performance of the ISA 50 and ISA 70 were found to be similar to Freund.s incomplete adjuvant for antibody production, but with less inflammatory response. The surfactant in ISA 50 and ISA 70 is a major component of the Freund.s adjuvant surfactant, mannide oleate.
Syntex Adjuvant Formulation (SAF)®
Recently developed as a Freund.s complete adjuvant alternative, this pre-formed oil-in-water emulsion is stabilized by Tween 80 and pluronic polyoxyethlene/olyoxypropylene block copolymer L121. SAF uses a low toxicity, high stimulatory derivative of muramyl dipeptide, thr-MDP, and the metabolizable oil squalene.
Antigen preparations should be free of extraneous microbial contamination. Millipore filtration of the antigen prior to mixing with the adjuvant is recommended.
The presence of byproducts, such as polyacrylamide gel, should be avoided because of their inflammatory or toxic properties.
Avoid pH extremes, particulate matter, and contamination with chemicals such as SDS, urea, acetic acid, or other solvents or potentially toxic agents.
Special precautions may be necessary if the antigen is a viable microbe.
If FCA is used, the mycobacteria should first be resuspended by vortexing or shaking. One part or less of Freund's adjuvant to one part antigen (v/v) is recommended. Two sterile luer-lock syringes, one containing Freund's adjuvant and one containing the antigen, preferably in sterile saline, are used for these purposes. Glass syringes or plastic disposable syringes without rubber plungers are preferred as the oil reacts with the rubber plunger on plastic disposable syringes. The antigen solution is injected into the adjuvant through a 3-way stopcock or mixing cannula, and the emulsion is prepared by pushing the solution back and forth between the syringes for several minutes. An emulsion is properly prepared when it becomes thick, is difficult to inject back and forth through the cannula, and will not separate on standing; a droplet placed into a saline solution should not disperse. Emulsification is enhanced by using cold (4 degrees C) adjuvant. Failure of the preparation to emulsify may be due to antigen contamination with SDS or organic solvents.
For antigen-adjuvant preparations using adjuvants other than Freund's, manufacturer's instructions must be followed.
Rabbits are the most commonly used laboratory animal species for polyclonal antibody production. Because of the inherent value of these experiments and the animals in which they are conducted, combined with the stress associated with these immunization protocols, specific-pathogen free rabbits must be used. These animals are readily available commercially and their use dramatically reduces the morbidity and mortality frequently documented in rabbits infected with microbial pathogens, especially Pasteurella multocida.
Rabbits are covered under the Animal Welfare Act (AWA), and, as such, all procedures pertaining to their use in research, teaching, and/or testing must be entered in the animal.s medical record. Failure to comply with the AWA will have significant negative consequences for UCLA. Information that must be recorded includes, but is not limited to, sedation/anesthetic events, blood collection, compound administration, and euthanasia. When administering any compound, the compound.s name, volume administered, and the site(s) where administered must be recorded. Rabbits maintained as part of long-term (>3 months) projects are expected to have periodic determinations of hematocrit as they are prone to developing anemia.
Use proper restraint methods during immunization and blood collection procedures to avoid animal and personnel injuries. Acclimating animals to handling and restraint procedures prior to the initiation of immunization or other experimental procedures reduces stress in animals and personnel. Those personnel who are uncomfortable or unfamiliar with animal restraint should contact DLAM for assistance. It is extremely important to train personnel to perform manual restraint techniques and to appropriately use commercially available restrainers. Rabbit sedation with acepromazine during immunization and blood collection reduces stress, enhances vasodilation, and can prevent injury to rabbits and personnel.
Immunization Site Selection, Preparation, and Administration
Carefully select and prepare the immunization site to preclude unnecessary pain and distress during handling and restraint and to minimize infection. Areas commonly used for physical restraint must not be immunized. These areas include the dorsal cervical/scapular and rump areas of rabbits, and the dorsal cervical/scapular regions and tail base in rodents. Skin injection sites must be at least 1-3 inches apart, depending on the animal's size.
The site must be prepared in a sterile manner to reduce the likelihood of abscess formation. Adequate preparation of the immunization field can significantly reduce the likelihood of negative consequences. Shave the area to be used and prep with three alternating scrubs of betadine and alcohol. Wear sterile gloves for injections. Change needles with every new injection site. Be sure to inject subcutaneously and not intradermally. Clipping the hair and properly preparing injection sites will reduce the potential for the development of infection or abscess formation and facilitate injection site visualization, thus permitting appropriate treatment of any developing immunization site lesions. The use of sterile needles and syringes is mandatory to minimize microbial contamination.
It is important that the staff administering Freund.s adjuvant be properly protected. Inadvertent needle sticks or other exposure can cause painful cutaneous lesions as well as cause the victim to be positive on future PPD tests. Proper PPE includes gloves and eye protection. It is especially important that the staff do not recap needles.
For injections, it is essential to use a needle with a sufficiently wide bore (gauge) to permit the smooth passage of the emulsion into the animal. Attempts to force the emulsion through a small needle gauge will result in separation of the needle from the syringe and .spattering. of the emulsion. Sterile glass syringes should be used as the rubber can interfere with the adjuvant.
Investigators proposing to use footpad and/or IP injections must provide scientific rationale to the ARC for review prior to performing these procedures as part of an approved ARC protocol.
Investigators or their staff must observe animals daily, including weekends and holidays, for the following: pain, swelling, abscessation, fistula formation, infection, and/or ulceration at or near the immunization site(s). This ensures timely and appropriate immunization site assessment and/or therapy. Investigators identifying animals with the above signs must contact a member of DLAM.s veterinary staff immediately (x42571). All observations must be entered into the animal.s medical record.
Generally, blood collection must follow the ARC Policy on Blood Collection from Laboratory Animals.
Specific blood collection concerns pertaining to polyclonal antibody production include:
Survival blood samples are generally collected via tail vein or retroorbital sinus from rodents under anesthesia and via the central ear artery in rabbits with appropriate sedation/tranquilization with acepromazine. Saphenous vein use in rodents does not require anesthesia. Isoflurane is commonly used for inhalant anesthesia in rodents, with precision vaporizers located throughout the various DLAM facilities.
Blood collection from rabbit ears by transecting the artery or vein is prohibited, as is rodent blood collection via tail transection or serial tail transection.
Xylene use is prohibited.
Because of the risk of cardiac tamponade, pulmonary hemorrhage, and pneumothorax, intracardiac blood collection is limited to terminal procedures and is performed under general anesthesia in both rodents and rabbits.
Enter the blood volume collected in the animal.s medical record, along with any hematocrit results.
Please contact DLAM should you need assistance with blood collection techniques as free training is available.
Antibody production in chickens is an alternative to the use of other animals for polyclonal antibody production. Antibody production in chickens offers the advantage of obtaining antibody through a non-invasive method (egg yolk).
Another alternative method in rabbits consists of placing a subcutaneous whiffle ball chamber. Immunizations are made directly into the chamber and antibody-rich fluid is harvested from the chamber. Advantages cited for this technique include greater flexibility in immunogen preparation, minimal discomfort and minimal tissue reaction, ease of immunization and fluid collection, and recovery of large volumes of antibody-rich fluid with low cellularity and absence of lipids. This procedure does require surgical placement of the chamber.
- Allison, AC; and Byars, NE. 1986. An adjuvant formulation that selectively elicits the formation of antibodies of protective isotypes and of cell-mediated immunity. J. Immunol. Methods 95:157-168.
- Allison, AC; and Byars, NE. 1991. Immunologic adjuvants: Desirable properties and side-effects. Mol. Immunol. 28(3):279-284.
- Altman, A.C, and Dixon, FJ. 1989. Immunomodifiers in vaccines. Adv.Vet. Sci. Comp. Med. 33:301-343.
- Amyx, HL. 1987. Control of animal pain and distress in antibody production and infectious disease studies. J. Am. Vet. Med. Assoc. 191(10):1287-1289.
- Bennett, B.; Check, IJ; Olsen MR; and Hunter, RJ. 1992. A comparison of commercially available adjuvants for use in research. J. Immunol.Methods 153(1-2):31-40.
- Bomford, R. 1988. Aluminum salts: Perspectives in their use as adjuvants. Pp. 35-41 in Immunological Adjuvants and Vaccines, G. Gregoriadis, A. Allison, and G. Poste, eds. New York: Plenum Press.
- Broderson, JR. 1989. A retrospective review of lesions associated with the use of Freund's adjuvant. Lab. Anim. Sci. 39:400-405.
- Brooks, RE; Betz, RD; and Moore, RD. 1978. Injury and repair of the lung: Response to intravenous Freund's adjuvant. J. Pathol. 124:205-217.
- Canadian Council on Animal Care (CCAC). 1993. CCAC Guidelines on acceptable immunological procedures. Ottawa, Ontario, Canada: CCAC (Available from CCAC, Constitution Square, Tower II, 315-350 Albert, Ottawa ON K1R 1B1. Tel: (613) 238-4031; Fax: (613) 238-2837; E-mail: email@example.com.)
- Check, IJ, Bennett, B; et al. 1990. Hunter's TiterMax®#R-1 Adjuvant: Technical Background. Norcross, Ga.: Vaxcel Corporation.
- Clemons, DJ; Besch-Williford, C; Steffen, EK; Riley,LK; and Moore, DH. 1992. Evaluation of a subcutaneously implanted chamber for antibody production in rabbits. Lab. Anim. Sci. 42(3):307-311.
- Croft, S; Walsh, RD; Lloyd, JW; and Russell-Jones, GJ. 1991. TraT: A powerful carrier molecule for the stimulation of immune responses to protein and peptide antigens. J. Immunol. 146:793-798.
- Ermeling, BL; Steffen, EK; Fish, RE and Hook, Jr, RR. 1992. Evaluation of subcutaneous chambers as an alternative to conventional methods of antibody production in chickens. Lab. Anim. Sci. 42(4):402-407.
- Grumpstrup-Scott, J; and Greenhouse, DD. 1988. NIH intramural recommendations for the research use of complete Freund's adjuvant. ILAR News 30(2):9.
- Hanly, WC; Artwohol, JE; Bennett. 1995. Review of Polyclonal Antibody Production Procedures in Mammals and Poultry. ILAR Journal 37(3): 93-118.
- Hillam, R.P, Tengerdy, RP and Brown, GL. 1974. Local antibody production against the murine toxin of Yersinia pestis in a golf-ball induced granuloma. Infect. Immun. 10:458-463.
- Harlow, E; and Lane, D. 1988. Adjuvants. Pp. 96-124 in Antibodies: A Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory.
- Herbert, W J. 1978. Mineral oil adjuvants and the immunization of laboratory animals. Pp. A2.3-A3.15 in Handbook of Experimental Immunology, D. M. Weir, ed.Oxford:Blackwell Scientific Publications.
- Hunter, R.L; Bennett, B; Howerton, D; Buynitzky, S and Check, IJ. 1989. Nonionic block copolymer surfactants as immunological adjuvants: Mechanisms of action and novel formulations. Pp. 133-144 in Immunological Adjuvants and Vaccines, G. Gregoriadis, A. C. Allison, and G. Poste, eds. New York: Plenum Press.
- Johnston, BA; Eisen, H; and Fry, D. 1991. An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits. Lab. Anim. Sci. 41:15-21.
- Karu, AE. 1993. Monoclonal antibodies and their use in detection of hazardous substances. Pp. 205-321 in Hazard Assessment of Chemicals-Current Developments, J. Saxena, ed. Washington, D.C.: Taylor & Francis Intl Publishers.
- Kleinman, NR; Kier, AB; Diaconu, D; and Lass, JH. 1993. Posterior paresis induced by Freund's adjuvant in guinea pigs. Lab. Anim. Sci. 43:364-366.
- Lacy, MJ and Voss, EW. 1986. A modified method to induce immune polyclonal ascites fluid in Balb/c mice using Sp2/0-Ag14 cells. J. Immunol. Methods. 97:169-177.
- Lipman, NS; Trudel, LJ; Murphy, JC; and Sahali, Y. 1992. Comparison of immune response potentiation and in vivo inflammatory effects of Freund's and Ribi adjuvants in mice. Lab. Anim. Sci. 42:193-197.
- Mahana, W; and Paraf, A. 1993. Mice ascites as a source of polyclonal and monoclonal antibodies. J. Immunol. Methods 167:187-192.
- Meyer, TJ; Ribi, EE; Azuma, I; and Zbar, B. 1974. Biologically active components from mycobacterial cell walls. II. Suppression and regression of strain-2 guinea pig hepatoma. J. Nat. Cancer Inst. 52:103-111.
- Nerenberg, ST; Zedler, P; Prasad, R; Biskup, NS; and Pedersen, L.1978. Hematological response of rabbits to chronic, repetitive, severe bleedings for the production of antisera. J. Immunol. Methods 24:19-24.
- Niemi, SM; Fox, JG; Brown, LR; and Langer, L. 1985. Evaluation of ethylene-vinyl acetate copolymer as a noninflammatory alternative to Freund's complete adjuvant in rabbits. Lab. Anim. Sci. 35:609-612.
- Ou, SK; Hwang, MC; and Patterson, PH. 1993. A modified method for obtaining large amounts of high-titer polyclonal ascites fluid. J. Immunol. Methods 165:75-80.
- Ribi, E; Cantrell, JL; Takayama, K; Qureshi, N; Peterson, J; and Ribi, HO. 1984. Lipid A and immunotherapy. Rev. Infect. Dis. 6:567-572.
- Schiefer, B; and Stunzi, H. 1979. Pulmonary lesions in guinea pigs and rats after subcutaneous injection of complete Freund's adjuvant or homologous pulmonary tissue. Zbl. Vet. Med. A. 26:1-10.
- Smith, DE; O'Brien, ME; Palmer, VJ; and Sadowski, JA. 1992. The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular weight antigen in rabbits. Lab. Anim. Sci. 42(6):599-601.
- Stills, HF; and Bailey, MQ. 1991. Use of Freund's complete adjuvant. Lab. Anim. 20(4):25-30.
- Stills, HF. 1994. Polyclonal antibody production. Pp. 435-448 in The Biology of the Laboratory Rabbit, 2nd Edition, P. J. Manning, D. H. Ringler, and C. E. Newcomer, eds. San Diego, Ca: Academic Press.
- Tillman, P; and Norman, C. 1983. Droperidol-fentanyl as an aid to blood collection in rabbits. Lab. Anim. Sci. 33(2):181-182.
- Toth, LA.; Dunlap, AW; Olson, GA; and Hessler, JR. 1989. An evaluation of distress following intraperitoneal immunization with Freund's Adjuvant in mice. Lab. Anim. Sci. 39(2):122-126.
- Toth, LA; and Olson, GA. 1990. Strategies for minimizing pain and distress in laboratory animals. Lab. Anim. 20(4):33-39.
- Yarkoni, E; and Rapp, HJ. 1979. Tumor regression after intralesional injection of mycobacterial components emulsified in 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene (squalene), 2,6,10,15,19, 23-hexamethyltetracosane (squalane), peanut oil, or mineral oil. Cancer Res. 39:1518-1520.
Approved 4/23/03; Revised 7/09/09