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.
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.
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.
Information regarding antibody availability can be found on
The Antibody Resource
in vivo production is sufficiently justified and approved
by the IACUC, the following standards for monoclonal antibodies
should be followed:
bleeding is strongly discouraged, but if used, should follow
the IACUC Standard Procedure (see below). For mice and rats,
of the tail clip is recommended.
Production: A maximum dose of 0.2 ml pristane is recommended.
No anesthetic is required for pristane priming.
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.
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
If complete Freund's adjuvant is used for the priming dose,
incomplete Freund's adjuvant should be used for the subsequent
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.
Collection: Maximum withdrawal should not exceed 5 ml/kg
per week or 10 ml/kg every other week for all species.
Sampling Routes: For rabbits, use ear vein; for mice, use
tail vein; for other species, use peripheral vessel.
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
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