Animal Care and Use

Adjuvants for Immunostimulation, Selection and Use of

IACUC Policy

Created prior to 01/2012
Revised 05/2015

PIs must consider, design, and use methods that lessen or eliminate pain and/or distress to enhance the well-being of animals used for experimental purposes. This includes the administration of adjuvants to animals to stimulate their immune response for research purposes.


Adjuvants produce an antigenic depot effect resulting in the sequestration and prolonged release of the antigen(s) of interest for the purpose of stimulating a prolonged antibody response. Unfortunately, this adjuvant-mediated immune stimulation may also result in an intense inflammatory response which causes pain and distress to the animals. Therefore, adjuvants must be used properly to minimize the pain and distress that may occur following their administration.


The PI is encouraged to use the information outlined below in designing immunization protocols that are most efficient in terms of the efficacy of antibody production and in terms of minimizing potential pain and distress for the animals used. The IACUC strongly encourages the use of a commercial vendor for rabbit antibody production. On site production of antibodies in the rabbit species requires scientific justification. Contact the Division of Comparative Medicine for suggested vendors.

Selected Adjuvants and their Properties

Freund’s Mineral Oil Adjuvant Emulsions. Freund’s complete adjuvant (FCA) is a mixture of non-metabolizable oil (mineral oil), a surfactant (mannide monooleate), and killed mycobacteria (Mycobacterium 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 immunologic 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 resulting in a prolonged antibody response. However, both can result in adverse reactions resulting from the stimulation of cell-mediated immune responses. FCA is particularly toxic when injected into laboratory animal species because of the host response to the non-metabolizable oil and the mycobacteria, which 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 thereby reducing the quantity of emulsion injected; c) multiple injection sites with limited 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 should be used only for weakly immunogenic antigens and only for initial immunizations. If booster immunizations are necessary, FIA should be used instead of FCA. The recommended volumes per injection site for Freund’s adjuvants are as follows:

Mouse0.05 ml0.10 ml0.10 ml
Rat/Other rodents0.05 ml0.10 ml0.20 ml
Rabbit0.10 ml0.20 ml0.50 ml
Larger Animal0.10 ml0.50 ml0.50 ml

Skin injection sites should be at least one to three inches apart, depending on the size of the animal, and should not be in areas used for handling and restraint. Footpad injections or IP injections with CFA are discouraged; however, they may be permitted in certain cases with adequate scientific justification.

Ribi Adjuvant Systems (RAS). The manufacturer (Ribi ImmunoChem Research, Inc., Hamilton, MT) has several mixtures of oil, detergent, and immunostimulator(s) to choose from. Three formulations of RAS, each containing different detoxified bacterial products as immunostimulators, are commercially available for either polyclonal or monoclonal antibody production: a) mycobacterial trehalose dimycolate (TDM) emulsion recommended for use with strong immunogens in mice, guinea pigs, and rats; b) gramnegative 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, goats, and other large animals. Adding aqueous antigen to the adjuvant and vortexing forms stable oil-in-water emulsions. In general, Ribi’s adjuvant emulsions, like other oil-in-water emulsions, are more suitable for protein antigens that are more hydrophobic or amphipathic than for proteins that are very hydrophilic since the effectiveness of RAS is dependent on the absorption of the antigen to the oil droplets in the oil-in-water emulsion. The inflammatory response associated with the use of these adjuvants is minimized by utilizing a metabolizable oil (squalene) and detoxified bacterial components as immunostimulators. Furthermore, since significantly less oil is needed to form stable oil-in-water emulsions than for stable water-in-oil emulsions (e.g., Freund’s-type adjuvants), a Ribi’s adjuvant should cause less tissue damage when injected into animals. It should be noted, however, that since RAS have 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 manufacturer of RAS are:

MPL + TDM Emulsion, R-700 or TDM Emulsion, R-760 (use with strong immunogens)

Mouse: 0.2 ml dose, administer 0.1 ml subcutaneously at two sites (Preferred)

or 0.2 ml intraperitoneally

Guinea Pig/Rat: 0.5 ml dose, administer 0.2 ml subcutaneously at two sites
and 0.1 ml intraperitoneally

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 production, boost intravenously (or intraperitoneally) with saline/antigen only 14 days after last adjuvant/antigen injection. Remove spleen for fusion three days after intravenous injection.

MPL + TDM + CWS Emulsion, R-730

Rabbit: 1.0 ml dose, administer 0.05 ml intradermally at six sites,
0.3 ml intramuscularly into each hind leg and 0.1 ml subcutaneously in neck region.

Goat: 1.0 ml dose, administer 0.5 intramuscularly into each hind leg

Inject on day 0, boost on day 28 and test bleed on day 38 to 42. If necessary, booster injections may be given every 28 days, using the same formulation. Note: Injecting more frequently than every 28 days may negatively impact results.

Hunter’s TiterMax (CytRx Corp., Norcross, GA). TiterMax is a water-in-oil emulsion, similar to Freund’s-type adjuvants, containing a metabolizable oil (squalene) and a nonionic surfactant (copolymer of polyoxyethylene and polyoxypropylene). Most of the adjuvant activity is attributed to the ability of the surfactant to activate and bind certain complement components, which putatively targets the antigen to follicular dendritic cells in the spleen and lymph nodes. Some studies suggest that TiterMax is superior to Freund’s-type 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:

 Injection RouteTotal InjectionsVolume per Injection
MouseIntramuscular20.02 ml
 Subcutaneous10.04 ml
RatIntramuscular20.05 ml
Guinea PigIntramuscular20.05 ml
 Subcutaneous40.05 ml
RabbitIntramuscular20.04 ml
 Subcutaneous40.10 ml
 Intradermal100.04 ml


  1. The Guide for the Care and Use of Laboratory Animals: 8th Edition. (Guide). National Research Council. 2011