Approved April 26, 2000

Monoclonal antibody production relies upon the use of animals for the harvesting of splenic B lymphocytes for the creation of hybridomas. Fusing with a myeloma cell line immortalizes these B cells for an inexhaustible in vitro supply of antibody. Currently there are no available sources for B lymphocytes other than live animals.

An alternative source of antibody from these hybridomas utilizes the growth in vivo of each cell line. Intraperitoneal injection of a hybridoma into a mouse or rat induces production of ascites fluid containing the monoclonal antibody. However, this method should only be considered when in vitro methods have not proven to be inadequate. Compliance with the following standards is necessary to minimize the discomfort or stress to which the animals may be subjected during the production of monoclonal antibodies.

1. If the creation of hybridoma cells will include the use of adjuvants for immunization of B lymphocyte donor mice/rats, the procedure must comply with the IACUC guidelines for immunizations, as detailed in "Immunization: Use of Freunds Adjuvant". The spleen is collected after the immunized animal is euthanized by a method which complies with the "AVMA Panel on Euthanasia" and institutional guidelines.

2. All cell lines used for hybridoma formation should be tested for the presence of rodent viruses prior to introduction into the animal colony. Any mice animal inoculated with untested or contaminated cell lines must be isolated in a special housing facility.

3. At present, Pristane is the most widely used agent for priming the peritoneal cavity. If Pristane is used to increase production of ascites fluid, the dose should not exceed 0.25 ml/animal. Other priming agents must be justified and reviewed on an individual basis by the IACUC.

4. After inoculation with an ascites-producing hybridoma line, animals must be carefully observed by the investigator every day to monitor the degree of abdominal distention and signs of illness.

5. The ascites fluid must be aspirated before abdominal distention causes respiratory distress or interferes with normal activity.

6. Skilled personnel may remove ascitic fluid with an 18 gauge or smaller needle without anesthesia. New personnel should be trained using anesthetized animals.

7. Animals must be humanely sacrificed according to approved euthanasia methods when taps are not productive, or the animal appears to be moribund as evidenced by lethargy, impaired mobility, or inability to reach food and water.

8. Ascites may be collected only three times from each animal before it is sacrificed, i.e., two survival taps and 1 terminal procedure. (The taps should be collected within 3 days of each other.)

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