Guidelines for Ascites Production in Mice:
1. In an attempt to avoid pain, discomfort, or distress in animals from the production of monoclonal antibodies (mAb) using mouse ascites, NIH in 1999 issued directives encouraging the use of alternative methods to produce monoclonal antibodies in vitro without compromising the aims of the study.
2. Institutional Animal Care and Use Committees are now expected to critically evaluate all protocols which propose using the mouse ascites method, and only allow in vivo production on the basis of strong scientific justification.
3. 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.
4. Based on Federal Guidelines, written documentation must be included in the IACUC protocol:
a. Scientific justification for the proposed use of the ascites method.
b. Why in vitro methods has been found to be unsuitable.
c. 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:
Description of procedures:
1. The volume of the priming agent must be reduced to as small a volume as necessary to elicit the growth of ascetic tumors and at the same time reduce the potential for distress caused by the irritant properties of the priming agent. Although 0.5 ml Pristane has been considered standard for adult mice, the lower dose of 0.1 -0.2 ml has been shown to be effective for many hybridomas.
2. Although the time 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 -0.5 ml and are usually 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.
3. Imported hybridomas must be MTBM (Molecular Testing of Biological Materials) analyzed before introduction into the animal host to prevent potential transmission of infectious agents from infected cell lines into facility mouse colonies and possibly to humans handling the animals.
4. 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.
5. 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 technique. The smallest needle possible that allows for good flow must be used (18 – 22 gauge or smaller.)
6. Wipe the abdomen with Betadine followed by 70% isopropyl alcohol. Withdraw ascites fluid through as small a needle as possible attached to syringe. Massaging the abdomen gently will facilitate fluid removal.
7. 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 2-3 ml warm saline or Lactated Ringer’s Solution (LRS) administered subcutaneously.
8. The number of taps must be limited, based on good body condition of the animal. A maximum of three survival taps (the 4th being terminal) is allowed.
9. 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) 2007. 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.