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Policy: Maintenance of Hybridomas in Rodents and the Collection of Ascites Fluid (IACUC)

Policy: Maintenance of Hybridomas in Rodents and the Collection of Ascites Fluid (IACUC)


The Institutional Animal Care and Use Committee (IACUC) has developed the following policy intended to eliminate or reduce to a minimum animal discomfort associated with in vivo production of monoclonal antibodies. When proposing to use the rodent ascites method of monoclonal antibody production, investigators must include the following information in the animal care and use protocol:

(a) confirmation that the procedures to be used may cause discomfort, distress or pain,
(b) description of the search for alternative methods, and
(c) scientific justification of the need for using the in vivo method including an explanation of why alternative in vitro methods cannot be used.

Externally generated immortal cell lines and rodent derived biologics that are to be used in hybridoma formation should be tested for presence of rodent viruses prior to introduction into the animal colony. The volume of pristane used to prime mice for ascites production should be minimized and should never exceed 0.5mL. After inoculation with an ascites-producing tumor line, rodents should be observed by the research staff at least three times per week for the first week and daily thereafter specifically to monitor degree of abdominal distention and signs of illness. Ascites fluid should be removed by tapping the peritoneal cavity before abdominal distention is great enough to cause discomfort or interference with normal activity. Mice should be euthanized if they show signs of poor condition such as huddling and ruffled coat.

The number of abdominal taps are to be limited to a maximum of three, with the last collection being a terminal procedure.

Removal of peritoneal fluid with a sterile 18 gauge or smaller needle may be done without anesthesia by skilled personnel. New personnel or students should be trained using anesthetized mice.


Monoclonal antibodies represent a powerful research tool, and the use of animals is indispensable to the establishment of monoclonal antibody producing cell lines. Once these cell lines are established, in vivo and/or in vitro techniques can be utilized for production of necessary quantity of monoclonal antibodies. There is evidence that the rodent ascites method of monoclonal antibody production causes discomfort, distress, and/or pain, and practical in vitro methods exist which can replace the ascites method in many experimental applications. In vitro methods should be used whenever feasible. There is, however, scientific justification for using in vivo methods for producing monoclonal antibodies in certain situations.


Role of the Investigator: The Investigator is responsible for justifying the appropriate methods to be used on a project-by-project basis and approved prior to initiation of project work.

Role of the IACUC: Federal regulations require the IACUC to critically evaluate proposed use of the mouse ascites method of monoclonal antibody production on a project-by-project basis. Prior to approval of proposals, which include this method, the IACUC must determine that (i) the proposed use is scientifically justified, (ii) methods that avoid or minimize discomfort, distress and pain (including in vitro methods) have been considered, and (iii) the latter have been found unsuitable. The IACUC fulfills this evaluation during the critical review of newly submitted animal care and use protocols and of amendments to previously approved protocols.


Guide for the Care and Use of Laboratory Animals, 8th Edition, NRC, 2011

Guidance Concerning the Production of Monoclonal Antibodies in Animals, OPRR, Notice: OD- 00-019, February 3, 2000

Monoclonal Antibody Production Report, National Research Council, 1999

Production of Monoclonal Antibodies Using Mouse Ascites Method, OPRR Reports, Number 98-01, November 17, 1997

Approved: 12/7/2010
Updated: 5/2/2017


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