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In Vivo Monoclonal Antibody Production Guidelines

Monoclonal antibodies (Mabs) have been established as exceptionally powerful research agents with potential for clinical use. Animal use (mouse) involved in the generation and batch production of Mabs involves immunization with and without testing for antibody production, euthanasia for lymphoid cell harvesting, and production of Mabs as ascites fluid. Alternative in vitro procedures for Mab production have been developed and the IACUC strongly encourages in vitro production in lieu of ascites production. However, it is recognized that ascites production of Mabs in vivo may be necessary in certain circumstances. The Mab producer must justify to the IACUC why in vitro methods are not adequate. The IACUC considers the production of in vivo Mab production to be a category E protocol (one that causes distress or discomfort that will not be alleviated).

Listed below are the IACUC guidelines for in vivo Mab generation and production. These guidelines are based on procedures known or assumed to decrease pain and distress. Deviations from these guidelines must be justified.

Immunization and Screening

Use of Complete Freund’s Adjuvant not conforming with the IACUC guidelines for this adjuvant must be justified. Blood sampling for detection of specific antibody production (screening) performed by retro-orbital puncture must be performed with animals under anesthesia. Blood may be collected from the mandibular/facial vein without anesthesia.


Methods for euthanasia not complying with the “AVMA Guidelines on Euthanasia” must be justified and approved by the IACUC.

Priming and Ascites Production

Pristane, the most commonly used priming agent for ascites production, is believed to act by inducing a granulomatous reaction and by interfering with peritoneal fluid drainage. A dose of 0.5 ml IP causes noticeable distress (Amyx, 1987) which is decreased with equally efficacious doses of 0.1 to 0.2 ml (Broduer, 1984; Colwell, 1986; Kwan, 1980). Therefore, pristane priming must be performed with doses no greater than 0.2 ml. Priming with agents other than pristane must be justified and approved the IACUC.

Although the time interval between priming and inoculation of hybridoma cells as well as the number of cells in the inoculums are determine empirically, inocula generally range from 105-107 cells in volumes of 0.1-0.5 ml and are usually administered 10-14 days after priming. Generally, very high cell numbers are associated with greater mortality and < 1×105 cells may elicit fewer ascetic tumors. Cell suspensions must be prepared under sterile conditions in physiological solutions.

Imported hybridomas must be analyzed before introduction into the animal host to prevent potential transmission of infectious agents from contaminated cell lines into facility mouse colonies and possible to human handling the animals. Please contact the OLAC for testing procedures.

Ascites Production

Accumulation of fluid in the peritoneal cavity causes respiratory distress. In addition, ascites fluid volume in mice can exceed the total blood volume of the animal increasing physiological distress from removal of the fluid due to hypovolemia. To lessen the respiratory and physiological stress in ascites production of Mabs the following procedures must be observed.

  • The animals must be examined at least once daily, seven days a week, following the observable appearance of ascites.
  • Ascites pressure should be relieved before abdominal distension is great enough to cause discomfort or interfere with normal activity. (See euthanasia criteria)
  • Manual restraint or anesthesia may be used for tapping. Isoflurane or methoxyflurane anesthesia is strongly recommended for removal of ascites fluid from the abdomen.
  • An 18-22 gauge or smaller needle should be used to remove the ascitic fluid. The person performing this procedure must be adequately trained or supervised by someone proficient in the procedure.
  • Animal(s) should be monitored frequently over several hours following the tap to observe possible signs of shock due to fluid withdrawal. Pale eyes, ears and muzzle and breathing difficulties are indicative of circulatory shock. Shock may be prevented or treated with 2 -3 ml warm saline or lactated ringers administered subcutaneously.
  • The number of taps should be limited, based on good body condition of the animal. A maximum of three survival taps (the 4th being terminal) are recommended. Additional taps should have individual IACUC approval.
  • Criteria for euthanasia of mice being used for in vivo Mab production
    1. Gross abdominal distention is present causing respiratory distress.
    2. Skin of the abdomen is a grey-green color.
    3. Removal of abdominal fluid does not relieve abdominal distention (solid tumor present).
    4. Animal is lethargic, dehydrated, inappetent, maintains a hunched posture and ruffled hair coat.
    5. Animal is moribund or emaciated.


  1. Amyx H.L. 1987. Control of Animal Pain and Distress in Antibody Production and Infectious Disease Studies. J. Am. Vet. Med. Assoc. 191(10):1287-1289.
  2. Brodeur B.R. Tsang P1. and Larose, Y. 1984. Parameters Affecting Ascites Tumor Formation in Mice and Monoclonal Antibody Production. J.Immunol. Methods 86:239-241.
  3. Colwell D.E., Michalek, S.M. and McGhee, J.R., 1986. Method for Generating a High Frequency of Hybridomas Producing Monoclonal IgA Antibodies. Methods Enzymol. 121:42-51.
  4. Jackson L.R., Fox J.G., 1995. Institutional Policies and Guidelines on Adjuvants and Antibody Production. ILAR Journal 37(3): 141-152.
  5. Marx U., Embleton M.J., Fischer R., et al 1997. Monoclonal Antibody Production The Report and Recommendations of ECVAM Workshop 23. ATLA 25, 121-137.
  6. McGuill and Rowan, 1989. Refinement of Monoclonal Antibody Production and Animal Well-Being. ILAR News 31:(1)7-10.
  7. Behavioral, Clinical, and Physiological Analysis of Mice Used for Ascites Monoclonal Antibody Production. Norman C. Peterson. Comparative Medicine 50(5): 516-526, 2000.
  8. ILAR report on Monoclonal Antibody Production. A Report of the Committee on Methods of Producing Monoclonal Antibodies. Institute for Laboratory Animal Research, National Research Council. 1999.

IACUC approved 4/6/1999
Revision approved 8/5/2008