Introduction
Monoclonal antibodies (mAb) are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer. These antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the subject of study. The cell lines are produced by fusing B cells from the immunized animal with myeloma cells (Köler and Milstein 1975). To produce the desired mAb, the cells must be grown in either of two ways: by injection into the peritoneal cavity of a suitably prepared mouse (the in vivo, or mouse ascites, method) or by in vitro tissue culture. Further processing of the mouse ascitic fluid and of the tissue culture supernatant might be required to obtain mAb with the required purity and concentration. The mouse ascites method is generally familiar, well understood, and widely available in many laboratories; but the mice require careful watching to minimize the pain or distress induced by excessive accumulation of fluid in the abdomen or by invasion of the viscera. The in vitro tissue-culture method would be widely adopted if it were as familiar and well understood as the mouse ascites method and if it produced the required amount of antibody with every cell line; but in vitro methods have been expensive and time-consuming relative to the costs and time required by the mouse ascites method and often failed to produce the required amount of antibody even with skilled manipulation. Modern in vitro methods have increased the success rate to over 90% and have reduced costs.
The anticipated use of the mAb will determine the amount required (Marx and others 1997). Only small amounts of mAb (less than 0.1 g) are required for most research projects and many analytic purposes. Medium-scale quantities (0.1–1g) are used for production of diagnostic kits and reagents and for efficacy
testing of new mAb in animals. Large-scale production of mAb is defined, in this context, as over 1 g. These larger quantities are used for routine diagnostic procedures and for therapeutic purposes.
The use of monoclonal antibodies (mAb) in biomedical research has been and will continue to be important for the identification of proteins, carbohydrates, and nucleic acids. Their use has led to the elucidation of many molecules that control cell replication and differentiation, advancing our knowledge of the relationship between molecular structure and function. These advances is basic biologic sciences have improved our understanding of the host response to infectious-disease agents and toxins produced by these agents, to transplanted organs and tissues, to spontaneously transformed cells (tumors), and to endogenous antigens (involved in autoimmunity). In addition, the exquisite specificity of mAb allows them to be used in humans and animals for disease diagnosis and treatment. Under the appropriate conditions, mAb-producing hybridomas survive indefinitely, so continued production of mAb is associated with the use of fewer animals, especially when production involves the use of in vitro methods. Despite all those benefits associated with production of mAb with the mouse ascites method, it can be distressful to the host animal.
The U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training (IRAC 1983) states that "animals selected for the procedure should be of appropriate species and quality and the minimum number required to obtain valid results. Methods such as mathematical models, computer simulation, and in vitro biological systems should be considered. Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative." The Guide for the Care and Use of Laboratory Animals (NRC 1996, page 10) specifically addresses excessive tumor burden in animals and states, "occasionally, protocols include procedures that have not been previously encountered or that have the potential to cause pain or distress that cannot be reliably controlled.... Relevant objective information regarding the procedures and the purpose of the study should be sought from the literature, veterinarians, investigators and others knowledgeable about the effects in animals." The Public Health Service Policy on Humane Care and Use of Laboratory Animals (NIH 1996, page 7) requires IACUCs to ensure that approved protocols conform with the PHS requirement that "procedures with animals ... avoid or minimize discomfort, distress and pain to animals (in a way that is) consistent with sound research design.” It is therefore incumbent on the scientist to consider first the use of in vitro methods for the production of mAb. If in vitro production of mAb is not reasonable or practical, the scientist may request permission to use the mouse ascites method. However, "prior to approval of proposals which include the mouse ascites method, IACUCs must determine that (i) the proposed use is scientifically justified, (ii) methods that avoid or minimize discomfort, distress
