Monoclonal Antibody Production
Approved June 25, 1998 (Download)
Background: Mice provide a major means of producing monoclonal antibodies, a process that involves the intraperitoneal inoculation of hybridoma tumor cells in combination with an adjuvant. The antibody rich ascitic fluid that subsequently accumulates in the peritoneal cavity as a result of these inoculations is harvested by needle aspiration.
Adjuvants are inflammatory agents or irritants and probably the one that is most commonly used to enhance the production of monoclonal antibodies in mice is Pristane. Typically, hybridoma, or monoclonal antibody secreting tumor cells are injected into the peritoneal cavity of mice 10-14 days after a “priming” dose of Pristane has been given. Doses of Pristane used in adult mice have ranged up to 0.5 ml but 0.1 to 0.2 ml have been found to be effective. Hybridoma cells are usually given at doses averaging 1,000,000 cells. Concentrations of 100,000 cells or less generally elicit fewer ascitic tumors, produce less ascitic fluid, and are far less likely to be effective.
The severity of the reaction and the amount of ascitic fluid produced is related to the doses of the adjuvant and hybridoma cells administered. The increased volume and pressure resulting from the accumulations of neoplastic cells and ascitic fluid can impair breathing and cause asphyxia if monitoring and removal of fluid is inadequate. Excessive quantities of fluid in the peritoneal cavity may be painful and shock may occur from the sudden decrease in fluid volume following harvest. The number of times such withdrawals should be performed should accordingly be minimized. It is customary to limit withdrawals to a total of two taps, unless the investigator provides evidence that the hybridoma is slow growing and additional taps can be accomplished in a humane fashion.
Policy: An In vitro method, such as culturing hybridomas in hollow fiber bioreactors or semipermeable plastic bags are being developed and offer an alternative to in vivo ascites production in some cases. Assurances must be provided to the IACUC that use of such alternatives has been tried and not been successful before approval for ascites production in animals will be granted.
The volume of priming agents such as Pristane should not exceed 0.2 ml in mice unless specifically justified. Cell suspensions must be in sterile physiologic solutions and the inoculations must be aseptically performed.
In addition to routine monitoring provided by the animal care staff, provisions must be described for adequately monitoring and treating inoculated mice by investigative staff. Monitoring of inoculated animals must be done by investigative staff at least three times a week for the first week and daily thereafter. Monitoring personnel must be trained and experienced in recognizing clinical signs associated with problems.
Ascitic fluid must be aseptically withdrawn before abdominal distension becomes great enough to interfere with normal activity. In no case should such distension be allowed to increase more than 20% beyond the normal diameter of the abdomen. While use of an anesthetic is preferred, manual restraint may be used, particularly in cases where anesthetic risk is increased. The smallest needle that allows for good flow should be used; needles larger than 18 gauge should not be used. The number of taps may not exceed a single survival tap and a second terminal tap, unless justified in the protocol and specifically approved by the IACUC.
Following survival taps, animals must be monitored and treated for shock if indicated. The responsible veterinary services staff veterinarian will immediately treat or euthanize any animal found by animal care or veterinary services personnel to be in respiratory distress or shock due to what is determined to be an excessive accumulation of ascitic fluid.
Animals should be euthanized appropriately before the final tap or at any point if there are signs of uncorrectible debilitation, pain or suffering. Signs can include hunched posture, rough hair coat, reduced food consumption, emaciation, inactivity, difficulty in ambulation, or respiratory problems.