BU IACUCInstitutional Animal Care and Use Committee IACUC oversee... approved April 2009, Revised January 2014, Revised July 2019

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 (PIPrincipal Investigator View Boston University's policy on...) 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.

Neisserial porins
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.
Repeat once.

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.





Routes of Administration Presenting Special Issues



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.


Other Considerations

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.

SpeciesTotal VolumeMaximum Volume per Injection Site
Total Volume LimitSubcutaneous SC (ml)Intradermal ID (ml)Intraperitoneal IPd(ml)Intramuscular IMb(ml)Footpad (ml)
Mouse<0.3ml<0.1<0.05 a=”” b=”” td=””><0.2b<0.05b,c0.05a,b,c

(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





Adopted from ARAC/NIH

BU IACUC approved April 2009, Revised January 2014