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 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 are under development and the IACUC encourages in vitro production in lieu of ascites; however, it is recognized that ascites production of Mabs in vivo remains the best option currently available.

Listed below are the IACUC guidelines for 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.

A. 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 retroorbital puncture must be performed with animals under anesthesia.

B. Euthanasia

• Methods for euthansia not complying with the "AVMA Panel on Euthanasia" must be justified.

C. 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 i.p. causes notiiceable distress (Amyx, 1987) which is decreased with equally efficacious doses of 0.1 to 0.2 ml (Brodeur, 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.

D. 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 due to hypovolemia. To lessen the respiratory and physiological stress in ascities production of Mabs the following procedures must be observed.
  1. The animals must be observed at least twice a day following the observable appearance of ascites.
  2. The ascites must be drained before respiratory distress is evident.
  3. The second collection must be within 3 days of the first and the animal must be euthanized immediately after this collection.
  4. Moribund animals must be euthanized by an approved method.

References

1. McGuill and Rowan, 1989. Refinement of Monoclonal Antibody Production and Animal Well-Being. ILAR News 31:(1)7-10.
2. 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.
3. Brodeur B.R. Tsang P1. and Larose, Y. 1984. Parameters Affecting Ascites Tumor Formation in Mice and Monooclonal Antibody Production. J.Immunol. Methods 86:239-241.
4. 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.