The consideration of alternatives to potentially painful procedures is required of investigators performing animal research at the University of Iowa. Freund's Complete Adjuvant has been the mainstay of immunological adjuvants in research for decades. While usually effective, the adjuvant may induce undesirable side effects which has lead to refinement of its use and to development of alternatives. To promote proper consideration by investigators of alternatives to Freund's Complete Adjuvant (FCA) the following list was compiled.

At this time there are no comprehensive or independent studies comparing a wide variety of these alternative adjuvants to each other. There are some limited studies which make some comparisons. These studies never look at all of these variables; they generally only compare a couple of adjuvants to each other and often not published in peer-reviewed journals.

This list is not comprehensive and does not represent endorsement of specific commercial products. However, the IACUC does endorse continual efforts to use refinements in animal research techniques and hopes that this information will be useful to investigators.

• Ribi Adjuvant System (RAS) - Ribi markets a wide variety of immunologic products. The primary product marketed as an alternative to Freund's complete adjuvant is a oil-in-water emulsion containing detoxified endotoxin (MPL) and mycobacterial cell wall components (TDW, CWS) in 2% squalene. It is as convenient to use as Freund's complete adjuvant (or more so because of its lower viscosity) and it has very low toxicity, having been used in humans. In some circumstances, especially in mice, it compares well with Freund's complete adjuvant. Preparation of the adjuvant-antigen emulsion in a tissue grinder with a teflon pestle (Potter-Elvehjem type) is encouraged for optimal results but it is not essential. No longer being produced.  Limited quantities are available from: Sigma M6536
  
• TiterMax - A product marketed specifically as a replacement for Freund's complete adjuvant, TiterMax is a stable, metabolizable water-in-oil adjuvant, a single injection of which the manufacturer has shown to induce antibody titers greater than 2 injections of Freund's (Freund's complete adjuvant followed by Freund's incomplete adjuvant). Available from: CytRx Corporation 150 Technology Parkway Technology Park/Atlanta Norcross, Georgia 30092  (800)-345-2987
  
• Syntex Adjuvant Formulation (SAF)³ - Has been developed as an alternative to Freund's complete adjuvant. It is a performed oil-in-water emulsion stabilized by Tween 80 and pluronic polyoxyethlene/polyoxypropylene block copolymer L121. SAF activates complement by the alternate pathway and is said to bias the humoral immune response to IgG2a in the mouse. Like other oil-in-water adjuvants, it works better with proteins that have some hydrophobic aspect to promote their adherence to oil droplets. Available from Chiron Corporation, Emeryville, California.
  
• Freund's Incomplete Adjuvant (FIA) - the most inflammatory component of FCA, killed mycobacteria, is not included in FIA. FIA is routinely used for boosting immunizations subsequent to FCA. It can also be used for the initial immunization, particularly when a strong antigen is used or moderate antibody levels are sufficient. As with FCA, efficacy is dependent upon vigorous mixing of the adjuvant and antigen until a stable emulsion has formed. Available from: Numerous research product companies.
  
• ALUM - aluminum hydroxide; Al(OH)3. Aluminum hydroxide is a widely used adjuvant, especially in commercial products such as vaccines. It is very well suited for strong antigens. Many sources of aluminum hydroxide are available. Commercially available as Alhydrogel, Accurate Chemical & Scientific Co, Westbury, New York.
  
• SuperCarrier - this and some similar products are convenient kits for coupling haptens, such as peptides and small proteins, to larger carrier molecules to enhance immunogenicity. The coupled proteins can be combined with other adjuvants. Syntex Research 3401 Hillview Ave. P.O. Box 10850 Palo Alto, CA 94303
  
• Elvax 40W^1,2 - this is an ethylene-vinyl acetate copolymer. While the production process is somewhat involved, the resulting immune response can surpass that induced by Freund's complete adjuvant. Sold in 25 gram amounts, however small gratis amounts are available. Available from: DuPont Chemical Co. Wilmington, DE (800)-628-6208; (request Elvax customer representative)
  
• L-tyrosine - a co-precipitate of the amino acid and antigen has been shown to have excellent adjuvant properties, even surpassing Freund's complete adjuvant in some circumstances. While the co-precipitation procedure is not difficult, it requires some manipulation. The cost is negligible. Available from numerous chemical companies.
  
• Montanide - a manide-oleate compound that has been shown to produce antibody levels equivalent to Freund's complete adjuvant. Small aliquots are available upon request from the producer. Available from: ISA Seppic Fairfield, NJ
  
• AdjuPrime - this is a carbohydrate polymer marketed as an alternative to Freund's complete adjuvant. Independent studies comparing its efficacy to other adjuvants do not appear to be available. The carbohydrate is thought to create both a "depot" effect and an enhancement of the interaction between the antigen and antigen-presenting cells.
  
• Nitrocellulose-absorbed protein⁸- Nitrocellulose-absorbed protein will give a desirable slow release of antigen over a period of 2 weeks to 2 months. The nitrocellulose is inert and causes minimal inflammatory response.
  
• Gerbu adjuvant - This is an aqueous phase adjuvant that does not have a depot effect. It therefore requires frequent boosting to achieve a high-titer response, but produces minimal inflammatory response. Gerbu adjuvant is available from C-C Biotech, Poway, California.
  
• Immune-stimulating complexes (ISCOMS) - ISCOMS are Ag-modified saponin/cholesterol micelles that form stable cage-like structures. ISCOM-associated antigen molecules do not form a depot site but are transported to the draining lymph nodes. Quantities of antigen as low as 1 ug have elicited a significant immune response. ISCOMS can be successfully prepared in the laboratory⁹.

Guidelines for use of Freund's Complete Adjuvant (FCA) in Laboratory Animals

Many of the undesirable side effects of FCA can be reduced or eliminated through refinements in its use. Subcutaneous and intramuscular routes with separation between inoculation sites adequate to avoid coalescence are preferred. The inoculation should be kept to the minimum volume practical and should not exceed the following guidelines.

Freund's Complete adjuvant (FCA) is widely used in research and is one of the first adjuvants developed. However, there are a number of significant disadvantages to it's use. Freund's complete adjuvant causes local inflammatory lesions which can be quite severe and result in chronic granulomas, abscesses, and tissue sloughs. Injected into the footpad, it can cause chronic lameness and arthritis; injected intraperitoneally, it can cause peritonitis. The American Veterinary Medical Association views pain induced by inflammatory agents as "an unnecessary and unwanted side effect of the use of inflammatory agents, or as a result of poor technique, and should not be accepted as a normal event in an immunization procedure". Furthermore, accidental inoculation of personnel with FCA can result in sensitization to tuberculin as well as chronic, local inflammation which is poorly responsive to antibiotic therapy. Therefore alternative adjuvants should be considered and utilized if and when possible. If adequate alternatives to FCA are not feasible, many of the undesirable side effects can be reduced or eliminated by the use of appropriate routes of administration, adequate separation of injection sites, and the use of a small amount of inoculum per site, which are described in the following guidelines:

• Before using FCA, consider the use of the incomplete Freund's adjuvant or a different adjuvant.
  
• If FCA must be used, it should be limited to the initial immunizing dose. Use of two or more doses of FCA is rarely warranted and must be strongly justified with objective data. Two to three weeks should be the minimum period after initial immunization with CFA, before reimmunization with a different antigen-adjuvant combination.
  
• Proper preparation of the FCA-antigen emulsion will limit inflammation. The mycobacterial component of FCA should be resuspended before use by vortexing or shaking. One part or less of FCA to one part antigen (v/v) is recommended. An emulsion is properly prepared when it becomes thick, will not separate on standing and will not disperse when a droplet is placed in saline. Emulsification is enhanced by use of cold (4C) adjuvant. It has been suggested that formulations of FCA not exceeding 0.1 mg dry mycobacterial cell mass/ml should be used to limit inflammation⁷.
  
• Routes of administration:

  Rabbits: The Canadian Council on Animal Care considers routes other than subcutaneous or intramuscular highly questionable or unacceptable. Intradermal injections often result in skin sloughing. This route should be discouraged unless strongly justified. If the intradermal route is unavoidable, a maximum of 0.05 ml should be administered per site. For subcutaneous injections , the inoculum should be divided into fractions of no more than 0.1 ml per site and not exceed a total volume of 1 ml. Injection sites should be separated from each other widely enough to ensure continued blood supply to adjacent areas of skin and subcutis. Prior to injection the site should clipped and aseptically prepared. Anatomic sites used for grasping, handling, or restraint such as the dorsal cervical/scapular areas should be avoided. Intramuscular inoculum should be of a volume not greater than 0.5 ml for rabbits, administered by a single injection into a deep muscle mass. The area where the needle penetrates the skin should be prepared with appropriate antiseptics. Foot pad immunization is not acceptable in rabbits.

  Mice: Intraperitoneal administration has been used to obtain higher titered polyclonal or monoclonal antibodies. The volume should not exceed 0.2 ml. Peritonitis can be minimized by limiting the use of FCA to the initial immunizing dose. Subcutaneous injections should be limited to a total volume of 0.2 ml with no more than 0.1 ml administered per site.

  Where scientific justification is provided (i.e. isolation of draining lymph nodes is required), foot pad immunization may be performed. The quantity of FCA administered should be minimal and only one foot per experimental animal should be injected. Also, the animal should be housed on soft bedding rather than screens to prevent painful ambulation. The maximum volume allowed is 0.05 ml for mice and 0.10 ml for rats.

• The use of sterile needles and syringes is mandatory.
  
• Precautions should be taken to avoid the inadvertent exposure (especially injection) of laboratory personnel to FCA.. Use of gloves, safety glasses, and laboratory coats is prudent. To avoid inadvertent injection, luer lock syringes should be used, needles should not re-capped, anesthesia or sedation should be utilized, and the technician should be familiar with proper animal restraint techniques.
  
• Injection sites should be clean and free of debris and contamination which could result in local infection.
  
• Additive inflammatory effects due to use of nonsterile preparations, excessive vehicle pH, or the presence of byproducts of purification such as polyacrylamide gel, SDS, urea, acetic acid, or other solvents or potentially toxic agents should be avoided. Special precautions may be necessary if the antigen itself is a viable microbe.
  
• Animals should be observed at least 3 times per week for a minimum of 4 weeks post-injection. Excessive swelling, abscess or fistula formation, and infection or ulceration at immunization sites should be brought to the attention of the veterinary staff. The observations should be recorded by the investigative staff. Post-procedural forms are available from the Office of Animal Resources which can be utilized for this purpose.
  
• Approximately 1% of a rabbits body weight in blood can be removed every two weeks for antibody harvest. Proposals requiring phlebotomies more frequent than every two weeks will require additional monitoring procedures such as measurement of hematocrit and total protein.

References:

1-Niemi SM, et al. Evaluation of ethylene-vinyl acetate copolymer as a noninflammatory alternative to Freund's Complete adjuvant in rabbits. Lab Anim Science 35:609-612, 1985.
2-Silberstein FB, et al. Elvax 40P implants: sustained, local release of bioreactive molecules influencing mammary ductal development. Developmental Bio 93:272-278, 1982.
3-Allison AC, et al. An adjuvant formulation that selectively elicits the formation of antibodies of protective isotypes and cell-mediated immunity. J Immuno Methods 95:157-158, 1986.
4-Rubach JA. Molecularly engineered microbial immunostimulators; In: Technological Advances in Vaccine Development, Alan R. Liss, Inc. 1988; 433-454.
5-Johnston BA, Eisen H, Fry D. An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits. Lab Anim Sci 41:15-21, 1991.
6-Amyx HL. Control of animal pain and distress in antibody production and infectious disease studies. J Am Vet Med Assoc 191:1287-89, 1987.
7-Broderson JR. A retrospective review of lesions associated with the use of Freund's adjuvant. Lab. Anim. Sci. 39:400-405, 1989.
8-Nilsson BO, Larsson A. Inert carriers for immunization. Res. Immunol. 143:553-557, 1992.
9-Coligan LG et al. eds. Current Protocols in Immunology, New York: John Wiley & Sons, 1995.