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Guidelines
Suggested parameters and sets of instructions outlining best practices and standards for accomplishing specific animal care and use research duties.

This Copy Was Generated On: May 9, 2025

Guidelines on Adjuvant Use in Rats, and Mice

Unit for Laboratory Animal Medicine

| Approval Date:

May 2, 2025 12:00 am

This Document Has Been Recently Updated

Labs May Have Questions Research Staff Review Recommended

Summary of Changes

  • Change the title from ‘Guidelines on Adjuvant Use in Rabbits, Rats, and Mice’ to ‘Guidelines on Adjuvant Use in Rats, and Mice’ since the part on Rabbits is removed
  • General review, language clarification and updates to reflect current practice

Who is Impacted

  • Investigative personnel

Impact

  • It is strongly recommended that research/investigative personnel review the document.

This document outlines available adjuvants and select procedures and techniques recommended by ULAM Veterinarians for use in rats, and mice.

Responsibility

  1. Principal Investigator (PI) and investigative personnel.

Glossary Definitions

Adjuvant

Pharmacological or immunological agent that modifies the effect of other agents (e.g., drugs, vaccines) while having few if any direct effects when given by itself.

Immunologic Adjuvant

Agents that non-specifically increase immune responses to specific antigens that are weakly immunogenic.

Viscous Adjuvant

An adjuvant that includes oil in its makeup.

Aqueous Adjuvant

An adjuvant that uses water or physiologic saline as its background.

Procedures

1. Adjuvant Selection

  1. Consider several factors when choosing an adjuvant: the size and composition of the antigen of interest, the species of animal involved in the model, and the route of administration.
  2. Consider the toxicity of the antigen preparation due to contamination with endotoxins or chemical residues and to ensure appropriate pH adjusted within physiological limits.
  3. This document delineates basic guidance regarding adjuvant use. Consult with the literature or an immunologist to choose the most appropriate adjuvant.

2. Viscous Adjuvants

  1. Freund’s Complete Adjuvant (FCA): FCA is a water-in-oil emulsion of mineral oil, mannide monooleate (a surfactant), and heat-killed or avirulent Mycobacterium tuberculosis or M. butyricum. It is effective in potentiating cellular and humoral responses to immunogens. Its activity is a result of sustained release of antigens from the oily deposit and stimulation of local innate immune responses.
    1. The use of FCA is associated with necrotizing dermatitis. Refer to Freund’s Incomplete Adjuvant (FIA) for further guidelines.
  2. Freund’s Incomplete Adjuvant (FIA): the same as FCA minus the mycobacterial cells or cellular components and thus, is less effective than FCA in inducing high antibody titers and enhancing cell-mediated immunity.
    1. The use of FCA/FIA is associated with necrotizing dermatitis, tissue sloughing, localized injection site granulomas, as well as diffuse systemic granulomas secondary to migration of the oil emulsion. Because of the potential for pain and distress, it is recommended that an alternative to FCA/FIA be used if possible.
    2. If FCA/FIA is scientifically justified, a few guidelines must be followed.
      1. FCA cannot be used more than once in the same animal as doing so may cause severe tissue damage.
      2. FCA with mycobacterial concentrations <0.1 mg/ml are recommended although mycobacterial concentrations of up to 0.5 mg/ml may be used without the requirement to provide scientific justification.
      3. FCA is usually necessary only for the initial immunization and subsequent immunizations are completed with FIA.
      4. Unless otherwise scientifically justified, routes of administration, sites of administration, volumes, and concentrations must follow the guidelines for FCA/FIA in this document.
  3. TiterMax ®: a microparticulate water-in-oil emulsion that uses the metabolizable oil squalene. Silica particles stabilize the emulsion and enables the emulsion to carry a wide variety of antigens without using large amounts of toxic emulsifying agents thus, minimizing lesions at the injection site. TiterMax is reported to produce antibody titer levels similar to FCA/FIA through activation of the complement system and inducing increased expression of HMC class II.

3. Aqueous (Mineral-based) Adjuvants

  1. Aluminum salts are claimed to be particularly effective at eliciting an immune response with weakly immunogenic antigens. Granulomas are common when subcutaneous or intradermal routes are used.
    1. Aluminum hydroxide: alhydrogels are sterile aluminum hydroxide gels which are pyrogen-free, stable and have a high adsorptive capacity. At pH <9, alhydrogels are positively charged which then readily adsorb negatively charged molecules such as proteins at neutral pH.
    2. Aluminum phosphate
  2. Calcium phosphate.

4. Route of Administration

  1. Possible routes of adjuvant administration are subcutaneous (SC), intraperitoneal (IP), intravenous (IV), or intradermal (ID). The appropriate route for use depends on the adjuvant and the animal species. When using oil or viscous gel adjuvant (e.g., FCA) in mice or rats, SC is the recommended route. Viscous adjuvants should never be given IV due to the risk of thrombosis and pulmonary embolism. Aqueous adjuvants (or antigens without adjuvant) are usually given SC, IP, or IV to rats and mice. For any adjuvant preparation not listed on this document, refer to the manufacturer and literature for recommendations on route of administration.
    1. SC: Subcutaneous injections allow for easy monitoring of the injection site and use the antigen-processing dendritic cells in the dermis (Langerhans cells). Space multiple injections away from each other so the resulting inflammatory lesions do not coalesce or join into a larger affected area.
    2. IM: Do not use IM injections in mice and rats due to their limited muscle mass and the ability of the adjuvant to travel along fascial planes resulting in irritation and inflammation of nerves and vasculature.
    3. IP: Aqueous adjuvants can be administered intraperitoneally in rats and mice. However IP injections of viscous adjuvants have been reported to induce peritonitis, acute inflammatory reactions, and behavioral changes and are often deemed to be inappropriate.
    4. IV: Intravenous is the preferred route for initial administration of small, particulate immunogens in aqueous adjuvants. The rapid, systemic absorption quickly delivers the antigens to the lymphatic system. Viscous adjuvants such as FCA and FIA cannot be given IV, as they will cause pulmonary emboli and granuloma formation.
    5. Footpad: Not recommended and requires scientific justification for approval. When footpad injections are scientifically justified they should be limited to one hind paw and the animal must be housed on soft bedding to minimize any interference with ambulation.
    6. Intrasplenic: Not recommended and requires scientific justification for approval.
    7. Intra-lymph node: Not recommended: requires scientific justification for approval.
    8. Other alternate routes include aerosol, oral, and intranasal in a variety of species.

5. Site of Administration

  1. Prepare the antigen in an aseptic manner including mixing.
  2. Clip and aseptically prepare all subcutaneous injection sites in all animals using Betadine or Nolvasan. This will reduce the likelihood of abscess formation and infection as well as facilitate monitoring of the injection sites. Use sterile needles, syringes, and aseptic technique for all injections to minimize the risk of infection and abscess.
  3. Choose injection sites that do not interfere with handling or restraint of the animal, blood draws, or the animal’s ability to move or bear weight. When administering multiple concurrent injections, sites should be clustered but distant enough from each other to prevent merging of resultant lesions or infections, should they occur.

6. Age

  1. Age is important to consider depending on the outcome of the immunization. The immune response in young adults tends to be robust and less affected by previous immune challenges than older animals. It is recommended that rats and mice are at least 6 weeks old.

7. Blood Collection Volumes

  1. Please refer to Guidelines on Blood Collection.

8. Injection Volume

  1. There are no universal recommendations for volumes of adjuvant to be administered. The goal is to use the smallest volume possible in order to elicit the desired antibody response. Start with the smallest recommended volume and modify as needed. It may be possible to use less than the minimum recommended volume per injection. NIH volume recommendations for immunizations using FCA or FIA are list below in Table 1. Suggested volumes, sites, and numbers of initial injections are outlined in Tables 1, 2 and 3.
  2. For other questions regarding volumes, please refer to Guidelines on Administration of Substances to Laboratory Animals.
    1. Table 1: Recommended Volume (mL) of Freund’s Complete or Incomplete Adjuvant per site by Species and Route

      SQ ID IP Footpad IM *
      Mouse < 0.1 * < 0.2 < 0.05 ** < 0.05
      Rat < 0.1 < 0.05 ** < 0.5 < 0.1 ** < 0.1

      *Not recommended
      **Only when justified
      For footpad injection, more than one limb requires justification.

    2. Table 2: Recommendations of Volume (mL) and Number of Injections Per Route for Viscous Adjuvant Administration in Rats and Mice
      Rat Mouse
      Route Site Volume # Injections Site Volume # Injections
      SC Base of tail 0.2 1 Base of tail  0.1 1
      IM * * * * * *
      ID * * * * * *

      *Not recommended

    3. Table 3: Recommendations of Volume (mL) and Number of Injections Per Route for Aqueous or No Adjuvant Administration in Rats and Mice 
      Rabbit Rat Mouse
      Route Site Volume # Injections Site Volume # Injections Site Volume # Injections
      SC Over each shoulder and quadriceps 1.5 1 Base of tail 0.5 1 Base of tail  0.5 1
      IM Into each quadriceps 0.2 – 0.5 2 * * * * * *
      IP * * * Abdomen 5 1 Abdomen 1 1
      ID Dorsum 0.05 4 – 10 * * * * * *
      IV
      (soluble antigens only)      		 Saphenous, cephalic,
      or auricular vein      		 1.5 1 Tail vein 0.5 Tail vein 0.2 1

      * Not recommended

      Adapted and modified from Leenaars and Hendriksen, 2005; CCAC 2002

9. Boosters

  1. Consider booster injections when antibody production has plateaued or begun to decline. If the initial immunization was given with a viscous (depot forming) adjuvant, one should wait at least 4 weeks before administering a booster. If the first immunization was with an aqueous adjuvant, or no adjuvant, boosters can be given after 2-3 weeks. Titers should be checked after the first booster to see if subsequent boosters are needed. Do not use FCA as a booster, due to severe tissue reactions with more than one injection in an animal. Instead, FIA can be used for a booster. The maximum recommended number of boosters is 3. Adjuvants are not always necessary in a booster injection.
  2. As long as there is no (or an insignificantly sized) lesion, give booster injections  near to the initial site, but not so close that the resulting inflammatory lesions can coalesce into one lesion. This takes advantage of memory cells in the lymph node(s) associated with that anatomical region. If there is a local lesion due to the initial immunization, give the booster at a different location. Never give booster injections into a granuloma or swelling. Do not give boosters IV or IP due to a high risk for inducing anaphylactic shock. If the initial injection was IV or IP, boosters should be given SC.

10. Ascites Production of Monoclonal Antibodies

  1. Collection of monoclonal antibodies via ascites has largely been replaced by in vitro methods using hybridomas. The necessity for utilizing the ascites method must be scientifically justified and proof provided that in vitro methods have failed for all appropriate cell lines.
  2. The total number of abdominal taps (withdrawal of fluid with a needle) is recommended to be three or less.

References

  1. CCAC Canadian Council on Animal Care. 2002. Guidelines on: Antibody Production. Ottawa Ontario Canada: CCAC (http://www.ccac.ca/Documents/Standards/Guidelines/Antibody_production.pdf).
  2. Leenaars M and Hendriksen CFM. 2005. Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations. ILAR J 46:269-279.
  3. NIH National Institutes of Health. Revised 2022. Guidelines for the Research Use of Adjuvants. (https://oacu.oir.nih.gov/system/files/media/file/2025-04/b8_adjuvants.pdf)
  4. Petrovsky N, Aguilar JC. 2004. Vaccine adjuvants: current state and future trends. Immunology and cell biology 82: 488-496.
  5. Stills HF Jr. 2005. Adjuvants and antibody production: dispelling myths associated with Freund’s Complete and other adjuvants. ILAR J 46:280-293.
  6. Stills HF. 2000. Antigens, antibodies, and blood collection. In: Silverman J, Suckow MA, Murthy S, eds. The IACUC Handbook. 2014. Boca Raton FL: CRC Press. 447-459.

SPECIES: Mice / Rats

Questions?

Questions or concerns about the content of this document should be directed to the Unit for Laboratory Animal Medicine (ULAM) at (734) 764-0277 or [email protected].