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Monoclonal Antibody Production Via Ascites in Mice

Purpose

To establish IACUC expectations and requirements when producing monoclonal antibodies using the ascites method in mice.

Definitions

Adjuvant:
An inflammatory agent or irritant that enhances the immune response to a given compound
Ascites:
The accumulation of fluid in the peritoneal cavity
Hybridoma:
A hybrid cell generated by the fusion of an antibody-producing cell with a tumor cell and used to produce a specific monoclonal antibody in vitro or in vivo

Background

Mice historically provided a major means of producing monoclonal antibodies via 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.

Pristane is the adjuvant most commonly used to enhance the production of monoclonal antibodies in mice. Typically, hybridoma cells are injected into the peritoneal cavity of mice 10-14 days after a “priming” dose of Pristane has been given. Volumes of Pristane used in adult mice must be limited to 0.1 to 0.2 ml, a dose which has been shown to be effective and produce fewer side effects than larger volumes. 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, one survival and one terminal.

In vitro methods, such as culturing hybridomas in hollow fiber bioreactors or semipermeable plastic bags, have been developed and offer an alternative to in vivo ascites production in most cases.

Policy

As part of the IACUC application for in vivo production of a specific antibody via ascites, the investigator must:

  1. perform a search of the current literature to determine if alternative in vitro methods can be used.
  2. provide assurance that the antibody is not commercially available from an in vitro source.
  3. provide strong scientific justification for use of the in vivo method, including assurance that in vitro alternatives have been tried and have not been successful.

The volume of priming agents such as Pristane should not exceed 0.2 ml in mice. Hybridoma cell lines must be tested for the presence of infectious agents prior to inoculation. Cell suspensions are generally in the range of 105-107 cells in a volume of up to 0.5 mL, 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. Mice should be weighed at least once a week. Monitoring personnel must be trained and experienced in recognizing clinical signs associated with complications.

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 (approximating the normal appearance of a pregnant animal) or the body weight increase 20% or greater. 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.

Following survival taps, animals must be monitored and treated for shock if indicated. Pale eyes, ears, or paws, and breathing difficulties can be indicative of shock. The responsible 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 untreatable debilitation, pain, or suffering. Signs can include hunched posture, rough haircoat, reduced food consumption, emaciation, inactivity, difficulty in ambulation, or respiratory distress.​

References

  1. Behavioral, Clinical, and Physiological Analysis of Mice Used for Ascites Monoclonal Antibody Production. Norman C. Peterson. Comparative Medicine 50(5): 516-526, 2000.
  2. Canadian Council on Animal Care Guidelines on: Antibody Production. 2002. guidelines on: antibody production (PDF)
  3. Jackson LR, Fox JG. Institutional Policies and Guidelines on Adjuvants and Antibody Production.ILAR Journal Volume 37, Number 3, 141-152, 1995.
  4. ILAR report on Monoclonal Antibody Production. A Report of the Committee on Methods of Producing Monoclonal Antibodies. Institute for Laboratory Animal Research, National Research Council. 1999. Monoclonal Antibody Production (PDF)
  5. The Johns Hopkins Center for Alternatives to Animal Testing. The Center for Alternatives to Animal Testing
  6. Cell Culture Company (C3). Custom Cell Culture Services

Approval/Review Dates

Originally A​​​pproved: 06/25/1998
Last Reviewed/Revised by the IACUC: 10/21/2021


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