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A. Monoclonal Antibody Production

IACUC Alternatives to Ascites Production of Monoclonal Antibodies

In an attempt to avoid pain, discomfort, or distress in animals from the production of monoclonal antibodies using mouse ascites, both OPRR and the USDA are encouraging the use of alternative methods to produce monoclonal antibodies in vitro without compromising the aims of the study.

Institutional Animal Care User Committees are now expected to critically evaluate all protocols which propose using the mouse ascites method, and only allow in vivo production on the basis of strong scientific justification.

UCSF has responded to this by setting up a core laboratory within the Cell Culture Facility to produce in vitro antibodies for UCSF investigators. For specific information on how to obtain in vitro antibodies, please contact Sally Miramon, Technical Supervisor of the Cell Culture Facility, at 476-8830.

Additional Information regarding antibody availability can be found on The Antibody Resource Page.

If in vivo production is sufficiently justified and approved by the IACUC, the following standards for monoclonal antibodies should be followed:

Retro-orbital bleeding is strongly discouraged, but if used, should follow the IACUC Standard Procedure (see below). For mice and rats, use of the tail clip is recommended.

Ascites Production: A maximum dose of 0.2 ml pristane is recommended. No anesthetic is required for pristane priming.

Collection of Ascites Fluid: Animals must be observed daily following the hybridoma injection in order to ensure that excessive bloating and discomfort do not occur. Bloating can result in severe respiratory distress. When mice appear bloated, ascites fluid must be removed.

Tapping: Withdraw ascites fluid through a 25 gauge needle attached to syringe. With the mouse lying on its back, swab the abdomen with ethanol and insert the 25 gauge needle into the abdomen. Massaging the abdomen gently will facilitate fluid removal. The maximum frequency of taps is every other day. Brief inhalant general anesthesia is recommended. Animals are to be euthanized after the final tapping procedure, or earlier if the animal is experiencing excessive bloating and/or respiratory distress.

B. Polyclonal Antibody Production

Adjuvant: If complete Freund's adjuvant is used for the priming dose, incomplete Freund's adjuvant should be used for the subsequent booster injections.

Immunization: Injections should be subcutaneous or, in rodents, intraperitoneal. Choice of other, less used routes, such as foot pads, should be justified by the investigator. For multiple sites, not more than 0.1 ml per site should be used for rabbits, 0.5 ml for sheep and goats, and 0.05 ml for mice. Sites should be well separated to prevent consolidation of inflammatory responses.

Blood Collection: Maximum withdrawal should not exceed 5 ml/kg per week or 10 ml/kg every other week for all species.

Titer Sampling Routes: For rabbits, use ear vein; for mice, use tail vein; for other species, use peripheral vessel.

Vasodilation: Do not use xylene or other inflammatory agents as vasodilating agents. Radiant heat or warm water is the recommended alternative. Anesthesia is not required for titer sampling using recommended routes.

Monitoring: Investigators assume responsibility for ensuring their animals' health through adequate monitoring. If clinical problems arise, they must consult the veterinary staff for assistance in managing them, or alternatively euthanize their animals.