The use of adjuvants in animal research studies of basic and applied immunology requires careful consideration. The requirement for relatively nonspecific inflammation to elicit robust immunity obliges the investigator to balance the cost of potential, local, and/or systemic pain and/or distress of the research animal due to the inflammation with the presumed scientific benefit to be gained from the experiment. The validity and applicability of the scientific knowledge gained must be tempered with acknowledgment that the use of potent inflammatory agents, particularly Complete Freund’s Adjuvant (CFA), can result in side effects. Whenever possible, alternatives to CFA must be used.1, 8 However, use of CFA remains scientifically justified in many systems, especially in the induction of autoimmune disease models for which no comparable alternatives exist.9
Intervals between immunizations depend upon the research goal and the animal’s response. It may be necessary to administer frequent injections if the research goal is to create an animal model for autoimmune disease. In contrast, if the goal is to produce antibodies to harvest for other projects, the principal investigator (PI) is advised to monitor the antibody titer regularly (every two weeks or so), and adjust the immunization schedule accordingly. Finally, the immunization schedule and animal response may also be dependent upon the size and antigenicity of the antigen and on the adjuvant used. As a general rule, administration of CFA followed by boosters of Incomplete Freund’s Adjuvant (IFA) for antibody production are separated by 4–6 weeks. In summary, injections should be separated in time as much as is commensurate with the goal of immunization.
There are many commercially available immunologic adjuvants. Selection of specific adjuvants or types depends on whether they are to be used for research and antibody production or in vaccine development. Adjuvants for vaccine use only need to produce protective antibodies and good systemic memory while those for antiserum production need to rapidly induce high titer and high avidity antibodies. No single adjuvant is ideal for all purposes and all have advantages and disadvantages. Adjuvant use is generally accompanied by undesirable side effects of varying severity and duration. Research on new adjuvants focuses on substances which have minimal toxicity while retaining maximum immunostimulation. Investigators should always be aware of potential pain and distress associated with adjuvant use in laboratory animals.
It is beyond the scope of this policy to describe all adjuvants here. CFA should be the last resort and others tried first. Below are some that are currently used in animal studies by investigators at Boston University.
NM PorB (Neisseria meningitidis membrane protein) or porin Ag complex:
On days 1, 14, and 28, administer 0.05–0.2ml SC in the flank of mice.
Ribi adjuvant followed by IFA
At six–ten weeks old, mice are immunized subcutaneously with 10–200ug of the Ag protein emulsified in 200ul Ribi.
Four weeks later, mice are boosted with the same Ag protein mixture emulsified in IFA.
Mice are euthanized three–four days after the second immunization.
Administer 200ul of a 1:1 emulsion of TiterMax and the Ag IP in mice.
Mice are immunized three times at two-week intervals with the Ag in 100ul of IFA. Two weeks after the last immunization, mice are euthanized.
Alternatives to Freund’s Adjuvant (Not Exhaustive)
When consistent with the scientific objectives, adjuvants known to produce less intense inflammatory responses must be considered as alternatives to CFA. These may include other microorganism-derived compounds [monophosphoryl lipid A (MPL, or the synthetic RC259), muramyl dipeptides and tripeptides (MDP and MTP), and TDM (trehalose dimycolate), etc.], other emulsions [TiterMax, Montanides, EMULSIGENS, Syntex Adjuvant Formulation (SAF), MF-59, and Specol, etc.], saponins (Quil A, and QS-21), aluminum compounds (e.g., alum), cytokines and immunostimulatory nucleic acids (e.g., CpG oligonucleotides), liposomes, virus-like particles, polymeric microspheres (polylactide co-glycolides), nanoparticles, subcutaneously implanted chambers5, and others10, 11, 12. In many situations these alternatives are capable of eliciting cellular and humoral antibody responses sufficient for many scientific purposes with fewer side effects than those commonly seen with CFA. Information on alternative adjuvants is also available online (see Websites below).
Ribi Adjuvant System
Ribi adjuvants are oil-in-water emulsions where antigens are mixed with small volumes of a metabolizable oil (squalene) which are then emulsified with saline containing the surfactant Tween 80. This system also contains refined mycobacterial products (cord factor, cell wall skeleton) as immunostimulants and bacterial monophosphoryl lipid A. Three different species-oriented formulations of the adjuvant system are available. These adjuvants interact with membranes of immune cells resulting in cytokine induction, which enhances antigen uptake, processing, and presentation. This adjuvant system is much less toxic and less potent than CFA but generally induces satisfactory amounts of high-avidity antibodies against protein antigens.
TiterMax represents a newer generation of adjuvants that are less toxic and contain no biologically derived materials. It is based upon mixtures of surfactant acting, linear, blocks or chains of nonionic copolymers polyoxypropylene (POP) and polyoxyethylene (POE). These copolymers are less toxic than many other surfactant materials and have potent adjuvant properties which favor chemotaxis and complement activation and antibody production. TiterMax adjuvant forms a microparticulate water-in-oil emulsion with a copolymer and metabolizable squalene oil. The copolymer is coated with emulsion stabilizing silica particles which allows for incorporation of large amounts of a wide variety of antigenic materials. The adjuvant active copolymer forms hydrophilic surfaces, which activate complement, immune cells and increased expression of class II major histocompatability molecules on macrophages. TiterMax presents antigens in a highly concentrated form to the immune system, which often results in antibody titers comparable to or higher than CFA.
Complete Freund’s Adjuvant
CFA, a water-in-oil emulsion containing heat-killed mycobacteria or mycobacterial cell wall components, is an effective means of potentiating cellular and humoral antibody response to injected immunogens. Adjuvant activity is a result of stimulation of immune cells by mycobacterial components present in CFA, while sustained release of antigens from the oily deposit and stimulation of a local innate immune response resulting in enhanced adaptive immunity occurs with both CFA and IFA. An essential component of this response is an intense inflammatory reaction at the site of antigen deposition resulting from an influx of leukocytes and their interaction with antigens. The use of CFA is an important biologic resource for investigators, which must be used responsibly and with care to avoid or minimize the adverse effects of excessive inflammation. CFA may result in local inflammation and granulomatous reactions at the site of injection. CFA can also cause more significant side effects such as chronic inflammation, skin ulceration, local abscess, or tissue sloughing. Other complications observed following CFA use are diffuse systemic granulomas secondary to migration of the oil emulsion, adjuvant-related arthritis, and, very rarely, chronic wasting disease.
The following guidelines are directed toward the elimination or minimization of complications secondary to immunization with CFA. These procedures are applicable and are required for administration of other adjuvants as well. Utilization of: a) sterile technique in the preparation of antigen-adjuvant emulsions; b) aseptic preparation of the injection site; c) appropriate injection technique; d) appropriate routes and sites of administration; e) adequate separation of injection sites; and f) use of smaller volumes at each injection site have all proven efficacious in the elimination of post-immunization complications.
Antigen preparations must be sterile and, ideally, isotonic, pH neutral, and free of urea, acetic acid, and other toxic solvents. Antigens separated using polyacrylamide gels must be further purified whenever possible or the amount of polyacrylamide gel must be reduced by careful trimming, to minimize the amount of secondary inflammation/irritation from gel fragments. Millipore filtration of the antigen prior to mixing it with the adjuvant is recommended to remove as much extraneous microbial contamination as possible.
The mycobacteria in CFA is resuspended by vortexing or shaking the ampule or vial. The CFA is then removed from the ampule or vial using sterile techniques. Although approaches may vary, one part or less of CFA to one part aqueous antigen solution (v/v) has been recommended1. Care must be taken to prevent introducing bubbles of air when mixing the CFA/antigen emulsion.
Although formulations of CFA containing 0.5mg/ml mycobacterial concentration are commercially available and have been used successfully by many researchers, concentrations of <0.1mg/ml are recommended to minimize the inflammation and necrosis observed with higher concentrations2. Use of greater concentrations than commercially available are not recommended unless scientifically justified and approved by the IACUC. In addition, use of preparations containing disrupted mycobacterial cells rather than whole, intact bacilli may prove desirable because of the inability of the latter to be distinguished histologically from live, acid-fast cells.
For most applications, CFA is usually only necessary for the initial immunization, while IFA, which lacks mycobacteria, is the adjuvant of choice for subsequent immunizations. Successive immunizations with CFA must be scientifically justified and approved by the IACUC. CFAs containing either M. butyricum or M. tuberculosis H37Ra (an avirulent strain) are commercially available. Additional information about CFA use is available online (see Websites below).
Experience has demonstrated that the use of injection volumes, routes of administration, and sites appropriate for the species and size of the animal (see Table 1 below) produce favorable results while minimizing undesirable side effects3, 4. Some routes of injection may be less disruptive to the animal than other routes (e.g., subcutaneous injection vs. footpad administration). Whenever possible, the least invasive methodology required to accomplish the experimental goal must be utilized. Intradermal and footpad injections must be avoided unless scientifically justified. Intramuscular injections must be justified as well. Separation of multiple injection sites by a distance sufficient to avoid coalescence of inflammatory lesions, and a minimum of two weeks between subsequent inoculations are recommended. In addition to the route of administration, the site of injection must be chosen with care to avoid areas that may compromise the normal movement or handling of the animal (e.g., intradermal injections in the scruff of the neck of a rabbit).
Routes of Administration Presenting Special Issues
1) Footpad immunization
Utilizing the footpad for immunization of small rodents may be necessary in particular studies where the isolation of a draining lymph node, as a primary action site, is required. The well-being of subject animals must be addressed by procedures such as limiting the quantity of adjuvant-antigen solution injected into the footpad, the use of only one foot per experimental animal, and housing on soft bedding rather than screens. In instances where no specific justification is provided for footpad inoculation, this technique must not be used for routine immunization of rodents. “Other institutes at NIH have successfully shown that it is possible to obtain the same lymphatic drainage by subcutaneously injecting at the border of the hairline on the skin proximal to the footpad (lateral tarsal region), which is believed to avoid much of the pain, in part, because it is not a weight-bearing, highly sensitive structure.”13 Alternative sites with potential draining lymph node utility include the hock (popliteal lymph node13) and cervical sites (auricular lymph node14; superficial cervical lymph node15). If scientific justification is provided, the recommended maximum footpad injection volumes are 0.01–0.05 in mice and 0.10ml for rats1. Rabbits must not be immunized in their feet because they do not have a true footpad.
2) Peritoneal exudate
The production of rodent peritoneal exudate by the intraperitoneal administration of antigens and adjuvants is a widely recognized, valid scientific procedure for obtaining high-titer reagent. Undesirable side effects of painful abdominal distention and the resulting distress can be avoided by daily monitoring and relief of ascites pressure, or termination of the experiment. Intraperitoneal injections of CFA-antigen emulsions must normally be limited to less than 0.2ml in mice6. For guidelines on production of monoclonal antibodies in mice, please see the IACUC Policy on Ascites Production.
3) Intramuscular immunization
Intramuscular injections are usually made in the biceps femoris or quadriceps muscle mass. Care must be exercised to avoid adjacent nerves and blood vessels as well as fascial planes when injecting into a muscle bundle. Intramuscular injection is generally not recommended in rodents because of their limited muscle mass.
Post-Injection Observations and Treatments
Post-inoculation monitoring of animals for pain and distress or complications at the injection sites is essential and must be done daily for a minimum of four weeks or until all lesions have healed. Supportive therapy may include topical cleansing, antibiotics, and use of an analgesic. Although post-inoculation analgesics are not routinely required, if overt pain or distress is observed, the use of narcotic agonists, mixed agonist-antagonists, or other species-appropriate agents must be considered, taking into account the research objective. Steroidal or non-steroidal anti-inflammatory agents must be used with caution due to their direct impacts on immunological processes and are not recommended.
The size needle used must always be the smallest gauge compatible with the emulsion to be injected.
Handling of adjuvants that contain mycobacterial products can be an occupational hazard to laboratory personnel. Reports of accidental needle punctures in humans have been associated with clinical pain, inflammatory lesions, and abscess formation in tuberculin-positive individuals. Tuberculin-negative individuals have tested positive in subsequent tuberculin tests after accidental CFA exposure7. Safety glasses must be worn to avoid accidental splashing of CFA in the eyes. Sedation of the animal subject is recommended as a means to avoid self-injection by research staff.
Scientists preparing antigens for in vivo administration in conjunction with adjuvants must be aware of the potential presence of contaminating substances and other characteristics of the injectate which may have additive inflammatory effects. Judicious use of adjuvants may be negated by failure to consider sterility of preparations, excessive vehicle pH, or the presence of by-products of purification such as polyacrylamide gel fragments. Care will be taken to consider and eliminate additional inflammatory stimuli whenever possible.
Table 1: Recommended Volume of CFA-Antigen Emulsion (CFA-AE) per Site and Route of Administration. These injection volumes are recommended for other adjuvants as well.
|Species||Total Volume||Maximum Volume per Injection Site|
|Total Volume Limit||Subcutaneous SC (ml)||Intradermal ID (ml)||Intraperitoneal IPd(ml)||Intramuscular IMb(ml)||Footpad (ml)|
(a) Not recommended
(b) Only when justified
(c) Only one limb recommended without justification
(d) Intraperitoneal administration requires daily monitoring and relief of ascites pressure
(e) N/A: Not applicable/not allowed in rabbits
Animal Preparation & Adjuvant Administration
1) Sedation and preemptive analgesia. If only one injection will be performed, sedation may not be required. It is strongly recommended if multiple site injections will be performed. Preemptive analgesia may be administered at the discretion of the PI and is required for CFA.
Administer sedation according the IACUC Policy for Anesthesia and Analgesia.
Ketamine: 50–90mg/kg IP +
Acepromazine: 1.0–2.0mg/kg IP +
Buprenorphine: 0.05–0.1mg/kg SC
Ketamine: 40–60mg/kg IP +
Acepromazine: 1.0mg/kg IP +
Buprenorphine: 0.025–0.075mg/kg IP or SC
Ketamine: 25mg/kg IM +
Acepromazine: 0.75–1.0mg/kg IM +
Butorphanol: 0.2–0.4mg/kg IM or SC OR
Buprenorphine: 0.02–0.05mg/kg IM or SC
2) Aseptic preparation of the injection site. This procedure is identical to preparation for aseptic surgery.
After the animal is sedated:
a) Administer sterile ophthalmic ointment to the eyes.
b) Shave an area larger than the required area for injection. Avoid the scruff of the neck in both rodents and rabbits.
c) Aseptically prepare the shaved area with betadine or Nolvasan (chlorhexidine), starting in the center and working toward the periphery in a circular motion. Then clean the same area using 70% isopropyl alcohol, working from the center and toward the periphery. Repeat the skin preparation one time, using first the betadine or chlorhexidine, followed by the isopropanol as described above.
3) After adjuvant administration is completed, return the animal to a clean, warm cage. If sedated, observe periodically until the animal is upright and moving about the cage.
4) Administration of the adjuvant—personnel procedure
a) Wearing gloves, perform the injection of the adjuvant, making certain that distances between injection sites are adequate to avoid coalescing of inflammatory lesions.
b) Be very careful not to inject yourself.
c) Dispose of all adjuvants and needles as required by Environmental Health & Safety.
5) Post-adjuvant administration animal monitoring and documentation
It is the responsibility of the PI or designated research staff to monitor and, if indicated, treat animals post-inoculation. Post-inoculation monitoring of animals for pain and distress or complications at the injection sites is essential and must be done daily for a minimum of four weeks or until all lesions have healed. The following guidelines must be observed.
Daily observations and any treatments administered must be recorded and be available for review by the Boston University Animal Science Center (BU ASC) staff for the first 28 days after inoculation.
Rats and mice
Document daily checks on each rodent injected using the hyperlinked rodent post-procedure monitoring sheet.
Document daily checks and treatments in the animal medical record. The animal medical record must also document the procedure, what was injected, and the sedation and analgesia administered.
1. Jackson, L.R., and J.G. Fox. 1995. Institutional Policies and Guidelines on Adjuvants and Antibody Production. ILAR Journal 37(3):141–150.
2. Broderson, J. R. 1989. A Retrospective Review of Lesions Associated with the use of Freund’s Adjuvant. Lab. Anim. Sci. 39:400–405.
3. Grumpstrup-Scott, J., and D. D. Greenhouse. 1988. NIH Intramural Recommendations for the Research use of Complete Freund’s adjuvant. ILAR News 30(2):9.
4. Stills, H. F., and M. Q. Bailey. 1991. The use of Freund’s Complete Adjuvant. Lab Animal 20(4):25–31.
5. Clemons, D. J., C. Besch-Williford, E. K. Steffen, L. K. Riley, and D. H. Moore. 1992. Evaluation of Subcutaneously Implanted Chamber for Antibody Production in Rabbits. Lab. Anim. Sci. 42(3):307–311.
6. Toth, L. A., A. W. Dunlap, G. A. Olson, and J. R. Hessler. 1989. An Evaluation of Distress Following Intraperitoneal Immunization with Freund’s Adjuvant in Mice. Lab. Anim. Sci. 39(2):122–126.
7. Chapel, H. M., and August, P. J. 1976. Report of Nine Cases of Accidental Injury to Man with Freund’s Complete Adjuvant. Clin. Exp. Immunol. 24:538–541.
8. Stills H.F. 2005 Adjuvants and antibody production: dispelling the myths associated with Freund’s complete and other adjuvants. ILAR journal. 46(3): 280–293.
9. Billiau, A., and P. Matthys. 2001. Modes of action of Freund’s adjuvants in experimental models of autoimmune diseases. Journal of Leukocyte Biology 70(6) 849–860.
10. Vaccine Adjuvants: Preparation Methods and Research Protocols. O’Hagan, Derek, T. Humana Press, 2000. DOI: 10.1226/0896037355
11. Schmidt, C.S., W. J. W. Morrow, and N. A. Sheikh. 2007. Smart Adjuvants. Expert Rev. Vaccines 6(3): 391–400.
12. Aguilar, J. C., and E. G. Rodriguez. 2007. Vaccine adjuvants revisited. Vaccine 25: 3752-3762.
13. Kamala, T. 2007. Hock Immunization: A humane alternative to mouse footpad injections. J. Immunol. Methods 328: 204–214.
14. Nierkens, S. et al. (2004). Evaluation of the Use of Reporter Antigens in an Auricular Lymph Node Assay to Assess the Immunosensitizing Potential of Drugs. Toxicological Sciences 79: 90–97.
15. Weaver, J. L. et al. (2005). Evaluation of a Lymph Node Proliferation Assay for its Ability to Detect Pharmaceuticals with Potential to Cause Immune-Mediated Drug Reactions. Journal of Immunotoxicology 2(1): 11–20.
16. Canadian Council on Animal Care (CCAC). Guidelines on Antibody Production. 2002.
Adjuvants and Antibody Production
Adopted from ARAC/NIH