Purpose

These guidelines address the importance of using in vitro methods of antibody production, the scientific justification required if animals are proposed for use, as well as the care and use of mice bearing ascites tumors in instances when the in vivo method is scientifically justified.

Background

Monoclonal antibodies are exceptionally powerful research tools and have potential clinical uses. In recent years there has been an increased availability and use of tissue-culture systems for the generation of monoclonal antibodies.^1,2 The Guide for the Care and Use of Laboratory Animals and the PHS Policy on the Humane Care of Laboratory Animals requires that in vitro methods be considered prior to in vivo methods. The Institute of Laboratory Animal Research³ Executive Summary recommendations include.

1.  The routine use of in vitro methods should be used;

2.  The use of the mouse ascites method should not be banned; and

3.  When the mouse ascites method is used efforts are made to minimize pain and distress.

Policy

Alternative methods to in vivo production must be considered before any in vivo methods are approved. The use of in vivo methods (i.e. mouse ascites) requires scientific justification, for example:

1.  Failure of a cell line to adapt in vitro;

2.  Purification methods lead to denaturation or decreased antibody activity;

3.  Contamination of the cell line; and

4.  Inabiltiy of cell line to maintain production of monoclonal antibodies.

 

If an investigator is unable to provide adequate justification, the IACUC may permit simultaneous evaluation of in vivo and in vitro methods for a period of 12 months. At this time, the Principal Investigator must provide the IACUC with documentation that in vitro methods are not feasible.

Sensitization protocols vary, but the use of Complete Freund’s Adjuvant (CFA) may be used once during the immunization process. Further immunizations, should use Incomplete Freund’s Adjuvant (IFA) or the antigen alone. When sufficient antibody titers are reached mice are euthanized and the spleen removed for cell fusion.

Priming of the mouse peritoneal cavity using pristine must not exceed 0.2 ml, as higher doses cause noticeable distress. ^4,5,6.

Ascites production is initiated by injection of hybridoma cells into the peritoneal cavity. The development of ascites leading to abdominal distention results in discomfort and distress.⁷ The mice must be observed and the observations documented at least twice daily by the investigator. Mice must also be weighed at least every other day beginning the day after hybridoma injection. Ascites fluid must be collected before body weight becomes 20% greater than the weight obtained prior to the injection, the abdominal distention is greater than a typical pregnant mouse, the body condition score deteriorates, or if they are unable to reach food or water.

Ascites fluid collection is a one time collection procedure performed on euthanized mice. Multiple peritoneocentesis is not allowed because: ^7,8

 

1.  Mice with ascites show signs of pain and distress, including decreased activity, decreased feed consumption;

2.  Survival times decrease with additional peritoneocentesis;

3.  Ascites tumors disseminate with time;

4.  Removal of ascites fluid results in circulatory shock; and

5.  Body condition score deteriorates over time as tumor burden increase.

References

1.  Peterson, NC and Peavey JE. Comparison of in vitro monoclonal antibody production methods with an in vivo ascites production technique. Contemporary Topics in Laboratory Animal Science. 37(5): 61-66. 1998.

2.  Stang, BV, Wood PA, Reddington JJ, REddington GM and Heidel JR. Monclonal Antibody production in gas-permeable flexible flasks, using serum free media. Contemporary Topics in Laboratory Animal Science. 37(6):55-60. 1998.

3.  Institute of Laboratory Animal Research. Monoclonal Antibody Production. 1999.

4.  Amyx H.L. 1987. Control of Animal Pain and Distress in Antibody Production and Infectious Disease Studies. J. Am. Vet. Med. Assoc. 191(10):1287-1289.

5.  Brodeur B.R. Tsang P1. and Larose, Y. 1984. Parameters Affecting Ascites Tumor Formation in Mice and Monooclonal Antibody Production. J.Immunol. Methods 86:239-241.

6.  Colwell D.E., Michalek, S.M. and McGhee, J.R., 1986. Method for Generating a High Frequency of Hybridomas Producing Monoclonal IgA Antibodies. Methods Enzymol. 121:42-51.

7.  Jackson Lr, Trudel, LJ, Fox JG and Lipman NS. Monclonal antibody production in murine ascites I. clinical and pathological features. Laboratory Animal Science 49(1):70-80. 1999.

8.  Peterson N.C. 2000 Behavioral, Clinical, and Physiological Analysis of Mice Used for Ascites Monoclonal Antibody Production. Comparative medicine Vol 50 No 5 516-526

9.  Fallon, M. 2002. Working with the IACUC: Writing an animal Care Protocol. AALAS

10.  McGuill and Rowan, 1989. Refinement of Monoclonal Antibody Production and Animal Well-Being. ILAR News 31:(1)7-10.

Approved by IACUC: 5/08/97
Revised: 8/12/99, 9/30/04