The following guidelines have been approved by the University Institutional Animal Care and Use Committee to insure that animals used in the development of monoclonal antibodies experience minimal pain and distress, yet provide sufficient quantities of antibody for research use. Production of monoclonal antibodies utilizing intraperitoneal injection of hybridomas must be scientifically justified in the Animal Care and Use Review Form and the in vitro production of monoclonal antibodies must be considered^8-23. The preferred method of production of monoclonal antibodies is utilizing in vitro technologies, which is recommended by the National Institute of Health and the investigator is encouraged to investigate that possibility before requesting to produce ascitic fluid.

• Immunization volumes should not exceed 0.2 ml when administered subcutaneously or 0.5 ml when administered by intraperitoneal injection. In vitro immunization procedures have been reported to be beneficial when antigen is in limited supply and previous attempts at in vivo immunization have failed. Other advantages of in vitro immunization are the requirement for fewer animals and the immunization period is reduced to 4-5 days^4,5,6.
• Pristane priming: The volume of pristane should not exceed 0.2 ml. Freund's incomplete adjuvant has been demonstrated to be as good or better than pristane as a priming agent ^1,2,3.
• It is recommended that hybridomas be tested for the presence of adventitious viral and mycoplasma agents prior to use in animals. Viral contamination is common in murine leukemia and transplantable tumor specimens. Inoculation may result in infection of the animal and spread of disease through the vivarium. Also, lymphocytic choriomeningitis virus has been isolated from tumor cell lines which is capable of producing illness in humans⁷.
•  Abdominal Paracentesis:
  • It is recommended that the number of abdominal taps should be limited to two taps. The second tap should be performed after the mouse has been euthanized. However, in more aggressive cell lines which cause significant morbidity it may be necessary to limit taps to less than this and vice versa for cell lines that are more slowly progressive and cause minimal morbidity, more than two taps may be appropriate.
  • Ascites pressure should be relieved when visible abdominal distention becomes evident, and prior to the development of marked abdominal distention with associated clinical signs of pain or distress. The undesirable side effect of painful abdominal distention can be avoided by daily monitoring, relieving ascites pressure, or terminating the procedure.
  • If paracentesis does not relieve abdominal distention the abdomen of the mouse should be gently palpated to determine if distention is due to intra-abdominal solid tumor growth. Confirmation of intra-abdominal solid tumor growth warrants euthanasia of the animal.
  • To minimize bacterial contamination, preparation of the paracentesis site with an antiseptic is recommended.
  • It is recommended that a large gauge needle (20 to 18 gauge) be utilized for paracentesis. Ascitic fluid is often viscous and a large gauge will allow more rapid collection thereby reducing the period of restraint required.
  • Administration of replacement fluids (i.e. 1-2 ml of saline, subcutaneously) should be considered when large volumes of ascitic fluid are harvested.
  • Post-paracentesis mice should be monitored for 30 minutes for the development of circulatory shock. Roughened haircoat, hunched posture, inactivity, pallor of the ears and eyes, tachypnea and dyspnea are compatible with shock. The presence of these signs warrant fluid administration. Persistence of these signs warrants notification of the veterinary staff or euthanasia of the animal.
• Daily monitoring of each animal administered intraperitoneal hybridoma cells is required (including weekends and holidays). Personnel should look for hunched posture, roughened haircoat, anorexia, dehydration, weight loss, loss of body condition, inactivity, difficulty in ambulation, tachypnea, and dyspnea. Any of the preceding signs of distress warrant notification of the veterinary staff or euthanasia of the animal.

Adapted from "Institutional Policies and Guidelines on Adjuvants and Antibody Production"; Jackson LR, Fox JG. ILAR Journal Vol 37, #3, p141-52, 1995.

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3-Jones SL, Cos JC, Person JE. Increased monoclonal antibody ascites production in mice primed with Freund's incomplete adjuvant. J. Immunol. Methods 129:227-31, 1990.

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5-Hengartner H, Luzzati AL, Schreier M, Fusion of in vitro immunized lymphoid cells with X63Ag8. Curr. Top. Microbiol. Immunol. 81:92-99, 1978.

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20-Moro et al. Multiparametric analyses of hybridoma growth on cylinders in a packed-bed bioreactor system with internal aeration. Serum-supplementation and serum-free media comparison for Mab production. J. Immunol. Methods 176:67-77, 1994.

21-Hopkinson J. Hollow fiber cell culture systems for economical cell-product manufacturing. Bio/Technology 3:225-230, 1985.

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23-Jackson LR, Trudel LJ, Fox JG, Lipman NS. Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production. J. Immunol. Methods (in press)