Antibody Production in Live Animals
Monoclonal Antibodies – created by identical immune cells that are cloned from a unique parent cell
Polyclonal Antibodies – created by several different immune cells
Adjuvant – an agent that stimulates the immune system. Adjuvants can be inorganic (e.g. alum) or organic (e.g. Complete Freund’s Adjuvant)
Ascites – antibody-rich fluid produced in an animal injected (usually in the peritoneal cavity) with a specific type of cell. Excessive build-up of ascites is associated with considerable pain and distress for the animals.
Produced in vitro in the laboratory using hybrid cell lines (hybridomas) – no IACUC oversight needed.
Purchased “Off the Shelf” – Antibodies obtained by a vendor that creates them in vitro in large quantities and sells them commercially (no IACUC oversight needed)
Prepared by a Commercial Vendor - Researcher supplies a specific antigen to a commercial laboratory, which then produces the antibody using animals in their facility (under their own IACUC oversight). Commercial laboratory sends resulting antibodies to researcher. Requires submission of a completed Custom Antibody Production Agreement.
Produced in the laboratory – Research team produces its own antibodies using live animals. Requires UCI IACUC review and approval (full protocol) with strong scientific justification.
Use of the ascites method for production of antibodies in a live animal requires strong scientific justification and adherence to the following guidelines:
- The proposed antibody is not commercially available; and/or commercially available alternatives are not suitable for the research objectives. Justification for the in-house production of antibodies may not be based on cost or convenience.
- No more than 0.1 ml Pristane, or 0.25 ml Freund's Incomplete Adjuvant, may be used for peritoneal cavity priming.
- Animals must be observed at least three times per week the first week, and daily thereafter, including weekends and holidays, to monitor the degree of abdominal distension, to relieve it as needed, and to look for other signs of illness.
- Fluid should be removed before abdominal distension is great enough to cause discomfort, or labored breathing, or to interfere with normal activity.
- Animals must be anesthetized prior to peritoneal tap.
- Ascites fluid may be harvested only once with recovery of the animal. Subsequent harvesting shall be performed as a terminal procedure.
- Ascites fluid should not exceed 20% of the baseline body weight.
- Animals that become moribund, cachexic, or that are otherwise unable to obtain food and water must be euthanized immediately.
Additional Guidelines for the Use of Complete Freund’s Adjuvant (CFA):
- Complete Freund's adjuvant should only be used for the first antigen dose. Subsequent injections should be with incomplete Freund's.
- Footpad injections with complete Freund's adjuvant are not acceptable.
- Injections of complete Freund's adjuvant should be subcutaneous or intraperitoneal. Intradermal injections can cause skin ulceration and necrosis. Intramuscular injections may lead to temporary or permanent lameness. Intravenous injections have been known to produce pulmonary lipid embolism.
- The injection containing the adjuvant should be divided into fractions so that no more than 0.1 ml is injected per site (subcutaneously) in rabbits or no more than 0.05 ml in mice.
- The injection site shall be monitored regularly for evidence of severe inflammation, large granuloma or ulceration. The veterinary staff must be contacted if any of these symptoms occur.
- Animals must be observed for evidence of pain, distress or infection resulting from the injection. The veterinary staff must be contacted should any of these symptoms occur.
- The inoculum should be free of extraneous microbial contamination. Millipore filtration of the antigen prior to mixing with adjuvant is recommended.
- Injection sites should be clean and free of debris, but need not be aseptically prepared.