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Antibody Production: Use of Adjuvants

Revised October 2003

These recommendations will be subject to continuous updating and suggestions for improvement or additions will be gratefully accepted. In the event that any of these recommendations cause unacceptable limitations to a principal investigator's ability to conduct research, the IACUC will consider appropriate justifications for modifications. Such justifications should be included on the Applications to Use Live Vertebrate Animals in Research or Educational Programs.

Polyclonal Antibodies in Rabbits

Improper or unnecessary use of Freund's Complete Adjuvant may cause pain in animals. Ulceration of the overlying skin always occurs with intradermal injections. Disseminated granulomas have been reported after subcutaneous or IV injections. Humans accidentally injected have suffered long-term painful abscesses.

  1. Instead of using Freund's Complete Adjuvant, use a less irritating adjuvant such as Freund's incomplete Adjuvant, RIBI (phone 800-548-7424, Fax 406-363-6129), or TiterMax (phone 404-368-9500, Fax 404-368-0622). Use RIBI and TiterMax according to the manufacturer's recommendation. The use of polyacrylamide gel as an adjuvant is discouraged due to severe ulcerative lesions (large nodules with fistulas; hot to the touch) it can cause in the animals. Its use must be strongly justified and the animals must be strictly monitored.
  2. Freund's Complete Adjuvant (FCA) should be used only when small amounts of soluble immunogens are available and using another adjuvant is not possible. The use of FCA must be scientifically justified in the animal use application. Supply supporting literature references.
    1. If FCA is used, it may be used only for the first dose since repeated doses of FCA are painful. All booster doses should be with Incomplete Freund's Adjuvant. Booster doses should not be closer than one week, and preferably two or more weeks, from the previous dose.
    2. Use a maximum of 1:1 antigen to adjuvant.
    3. Limit the volume administered to only one of the following: (If multiple routes are to be used, do not exceed 2.0cc for the total dose)
    4. Investigator should contact the Division of Laboratory Animal Medicine for consultation if ulcerative or nodular lesions develop.
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Routes of Administration

All of the following doses assume a 1:1 mixture of antigen: FCA. Always use sterile equipment when mixing the antigen and adjuvant. Only sterile equipment should be used for injections and always take appropriate precautions to sanitize the injection sites.

  1. Intramuscular Route: Maximum total dose, 2.0cc with a maximum dose per site of 0.5cc. Use deep injections into large muscle masses such as those at the posterior aspect of the rear limb or adjacent to the lumbar vertebrae.
  2. Subcutaneous Route: Doses are the same as intramuscular. Sites can be along the back and lateral to the vertebral column or immediately behind the scapulae.
  3. Intradermal Route: Maximum total dose, 1.0cc with a maximum dose per site of 0.05cc (therefore, 20 sites). Site spacing should be at least 3.0cm and the area to be injected should be prepared as for aseptic surgery. Sites should be restricted to the skin of the back from the shoulders to the hips.
  4. Other Routes: The use of foot pad immunizations or the intraperitoneal and intravenous routes are prohibited.
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Additional Precautions

The use of antigens purified by various electrophoretic procedures has produced severe inflammatory reactions in rabbits. Therefore, the following precautions are recommended:

  1. All procedures used in the preparation of the end product must be as clean (sterile) as possible to avoid bacterial contamination. The hair should be clipped and the skin decontaminated with a surgical scrub.
  2. Every attempt should be made to purify the antigen by eliminating contamination by the polyacrylamide gel, DMSO, or any other eluting chemicals.
  3. When gels or nitrocellulose strips containing antigen must be used to immunize animals, reducing the particle sizes to facilitate their injection, as a suspension is superior to surgical implantation of entire strips.
  4. Excessive swelling, scratching, chewing, and self-mutilation of the injection site are signs of infection, pain or irritation. Any abnormalities should be reported to the Division of Laboratory Animal Medicine (DLAM) Veterinary Care Staff (966-3111).
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Mouse Ascites Production

NOTE: Creating ascites in mice for the production of monoclonal antibodies is no longer routinely acceptable. In order to use mice for the production of monoclonal antibodies, the Principal investigator must supply documentation and scientific justification that ascites cannot be produced by non-animal alternatives.

Monoclonal Antibodies in Mice

Ascites production must be done to minimize distress. The following are recommended:

  1. The use of pristane as an intraperitoneal priming agent should be limited to a single dose of no more than 0.1cc per animal.
  2. Animals developing ascites must be watched closely and have fluid drained before reaching a stage where locomotion is impaired or other signs of distress are present. Ascites accumulation should not exceed 10-15% of the initial body weight.
  3. Aseptic precautions should always be observed when fluid is withdrawn.
  4. It is recommended that the animal be allowed to survive only an initial tap of the accumulated fluid. The second collection should be made after the animal has been euthanized.
  5. In the case of solid tumors, every attempt should be made to harvest cells before the animal suffers noticeable impairment of normal functions, i.e., locomotion, eating, grooming, etc. Tumor size in mice should not exceed 1.3 cm.
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