Ascites Method for Antibody Production in Mice (IACUC)

In an attempt to avoid pain, discomfort, or distress in animals from the production of monoclonal antibodies (mAb) using mouse ascites, the NIH in 1999 issued directives encouraging the use of alternative methods to produce monoclonal antibodies in vitro without compromising the aims of the study.

Institutional Animal Care & Use Committees critically evaluate all protocols which propose using the mouse ascites method, and only allow in vivo production on the basis of strong scientific justification. Protocols using the Ascites method for production of monoclonal antibodies should be at minimum categorized as Category D as ascites production will results in more than momentary pain and distress. Analgesia must also be detailed, unless scientifically justified.

There are legitimate circumstances for the use of the ascites method (for example, in vitro methods fail to generate sufficient quantities of mAb for use). However, since practical in vitro methods for large-scale production are available, the use of the ascites method should be the exception. Adherence to this policy is mandatory unless a specific exception has been approved by the IACUC.

Based on federal guidelines, written documentation must be included in the IACUC protocol:

• Scientific justification for the proposed use of the ascites method.
• Why in vitro methods have been found to be unsuitable.
• A description of the methods that will be used in order to prevent or minimize pain and distress.

If in vivo production is sufficiently justified and approved by the IACUC, the following standards for monoclonal antibodies must be followed.

The volume of the priming agent must be reduced to as small a volume as necessary to elicit the growth of ascetic tumors and minimize the potential for distress caused by the irritant properties of the priming agent. Although 0.5ml pristane has been considered standard for adult mice, the lower dose of 0.1–0.2ml has been shown to be effective for many hybridomas.

The interval between priming and inoculation of hybridoma cells as well as the number of cells in the inoculum are determined empirically. Inocula usually range from 105–107 cells in volumes of 0.1–0.5ml administered 10–14 days after priming. Generally, very high cell numbers are associated with greater mortality and 1 x 105 cells may elicit fewer ascetic tumors. Cell suspensions must be prepared under sterile conditions in physiological solutions.

Imported hybridomas or other materials of biological origin must be screened for adventitious murine pathogens before introduction into the animal host to prevent potential transmission of infectious agents from infected cell lines or products into facility mouse colonies and possibly to humans handling the animals. Please see Pathogen Screening of Biological Materials Introduced into Rodents for more information on this topic as well as instructions on testing of these materials.

Animals must be monitored at least once daily, seven days a week, by personnel familiar with clinical signs associated with ascites and circulatory shock. Bloating can result in severe respiratory distress and hypovolemia with associated decreased blood pressure and shock.

Ascites pressure must be relieved before abdominal distension is great enough to cause discomfort or interfere with normal activity. Manual restraint or anesthesia may be used for tapping. The tap must be performed by trained personnel using proper aseptic techniques. The smallest possible needle that allows for good flow must be used (18–22 gauge or smaller).

Wipe the abdomen with Betadine followed by 70% isopropyl alcohol. Withdraw ascites fluid through as small a needle as possible attached to a syringe. Massaging the abdomen gently will facilitate fluid removal.

Animals must be monitored frequently over several hours following the tap to observe possible signs of shock due to fluid withdrawal. Pale eyes, ears, and muzzle and respiratory distress are indicative of circulatory shock. Shock may be prevented or treated with 1mL SQ and 1 mL IP of warm saline or LRS.

The number of taps must be limited to a maximum of three survival taps (the fourth being terminal). If at any point an animal meets euthanasia criteria during monoclonal antibody production, euthanasia should be performed regardless of if the animal has not met the maximum amount of taps.

Mice should be euthanized appropriately before the final tap or promptly if there is evidence of debilitation, pain, or distress. Signs of distress include hunched posture, rough hair coat, reduced food consumption, emaciation, inactivity, difficulty in ambulation, respiratory problems, and solid tumor growth.

Monoclonal Antibody Production. A Report of the Committee on Methods of Producing Monoclonal Antibodies. ILAR. NRC. 1999. http://grants.nih.gov/grants/policy/antibodies.pdf
Guidelines for Ascites Production in Mice. NIH Animal Research Advisory Committee (ARAC) 2016. http://oacu.od.nih.gov/ARAC/documents/Ascites.pdf‎
Peterson, N.C. Behavioral, Clinical, and Physiological Analysis of Mice Used for Ascites Monoclonal Antibody Production. Comparative Medicine 50(5):516-526, 2000.
ILAR Journal 37(3): 141-152, 1995.