These guidelines address the importance of using in vitro methods for 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.
Monoclonal antibodies are exceptionally powerful research tools and have potential clinical uses. There is increased availability and use of tissue-culture systems for the generation of monoclonal antibodies1,2. 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 the use of in vivo methods. The Institute of Laboratory Animal Research3 Executive Summary recommendations includes the following statements:
- The routine use of in vitro methods is preferred;
- The use of the mouse ascites method should not be banned; and
- When the mouse ascites method is used efforts are made to minimize pain and distress.
Alternative methods, rather than 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, which may include, for example:
- Failure of a cell line to adapt to in vitro culture.
- Purification methods lead to denaturation or decreased antibody activity.
- Contamination and resulting loss of utility of the cell line.
- Inability of the cell line to maintain production of monoclonal antibodies.
If an investigator is unable to provide adequate justification for in vivo use, the IACUC may permit simultaneous evaluation of in vivo and in vitro methods for a period of approximately12 months. By the end of this period, the Principal Investigator must provide the IACUC with convincing documentation that production of the desired antibody using the in vitro methods will not be feasible.
If a researcher prefers to use an off-campus organization for the production of custom antibodies in animals, that organization must have an Animal Welfare Assurance on file with NIH/Office of Laboratory Animal Welfare (OLAW). Antibodies are considered customized if produced using antigen(s) provided by or at the request of the investigator (i.e., not purchased “off- the-shelf”).
Sensitization protocols may vary, however Complete Freund’s Adjuvant (CFA) may be used only once during the immunization process. Further immunizations must 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 prior to injection of hybridoma cells using pristane must not exceed 0.2 ml, as higher doses cause noticeable distress4-6.
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 distress7. The mice must be observed and the observations documented a minimum of twice daily by the investigator. Mice must be weighed prior to injection and a minimum of 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 mice are unable to reach food or water.
Ascites fluid collection is a one time collection procedure performed on mice that haven been euthanized. Multiple peritoneocentesis is not allowed because 7,8:
- Mice with ascites show signs of pain and distress, including decreased activity, and decreased feed consumption.
- Survival times decrease with additional peritoneocentesis.
- Ascites tumors disseminate with time.
- Removal of ascites fluid results in circulatory shock.
- Body condition score deteriorates over time as tumor burden increases.
- Peterson, NC and Peavey JE. Comparison of in vitro monoclonal antibody production methods with an in vivo ascites production technique. Contemp Top Lab Anim Sci. 37:61-66. 1998.
- Stang, BV, Wood PA, Reddington JJ, REddington GM and Heidel JR. Monoclonal Antibody production in gas-permeable flexible flasks, using serum free media. Contemp Top Lab Anim Sci. 37:55-60. 1998.
- Institute of Laboratory Animal Research. Monoclonal Antibody Production. 1999.
- Amyx HL. Control of Animal Pain and Distress in Antibody Production and Infectious Disease Studies. J. Am Vet Med Assoc 191:1287-1289, 1987.
- Brodeur BR, Tsang PI, and Larose, Y. Parameters affecting ascites tumor formation in mice and monoclonal antibody production. J Immunol Methods 86:239-241, 1984.
- Colwell DE, Michalek SM, and McGhee JR. Method for generating a high frequency of hybridomas producing monoclonal IgA antibodies. Methods Enzymol. 121:42-51, 1986.
- Jackson LR, Trudel LJ, Fox JG, and Lipman NS. Monoclonal antibody production in murine ascites I. Clinical and pathological features. Lab Anim Sci 49:70-80. 1999.
- Peterson NC. Behavioral, clinical, and physiological analysis of mice used for ascites monoclonal antibody production. Comp Med 50:516-526, 2000.
- Working with the IACUC: non-VA version AALAS Learning Library, 2012.
- McGuill MW and Rowan AN. Refinement of monoclonal antibody production and animal well-being. ILAR J 31:7-10, 1989.
Approved by IACUC: 06/16/16
Enabled by: OLAW