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Stop Animal Exploitation NOW!
S. A. E. N.
"Exposing the truth to wipe out animal experimentation"

Articles and Reports

Alternatives to Animal Research and Testing

Alternatives to Ascites Production of Monoclonal Antibodies
by John McArdle, Ph.D.
Alternatives Research and Development Foundation
Eden Prairie, Minnesota
www.nal.usda.gov/awic/newsletters/v8n3/8n3mcard.htm

Pros And Cons: Ascites vs. In Vitro

Support for continued use of ascites methods are usually based on claims of: (1) more rapid production and high yields of highly concentrated monoclonal antibodies; (2) minimal requirements for materials, labor, and technical expertise; (3) most hybridomas will grow in mice; (4) relatively less expensive; and (5) either an inherit resistance to or lack of familiarity with in vitro methods.

Experience in European laboratories strongly suggests that "these arguments are increasingly being challenged by documents showing that, depending on the amount and concentration of MAbs needed, in vitro production methods are equally well suited"as ascites (Hendriksen, personal communication). With continually increasing expenses associated with the care of laboratory animals and decreasing prices associated with either the newer in vitro methods or decreased production costs for existing in vitro equipment, the cost differential between animal- and non-animal-based methods is rapidly disappearing.

In contrast with in vivo methods, in vitro approaches to monoclonal antibody production have many positive attributes. In general, MAbs derived from in vitro alternatives express immunoreactivity in ranges of 90 to 95 percent regardless of the method used. This is significantly higher than that with MAbs produced by ascites. Similarly, in vitro cultures rarely fail (3 percent or less), while a much higher percentage of ascitic mice do not produce antibodies. Further, the quality of in vitro MAbs is equal to or better than that derived from in vivo methods.

Glycosylation has been raised as an issue with in vitro methods for producing Mabs. (Ed. note: Glycosylation can influence the antigen-binding capacity, the resistance of an antibody to proteolysis, and other biologically important processes.) However, glycosylation patterns are more easily regulated in vitro, since with ascites, they can vary between each individual animal. The ECVAM committee concluded that "there are no reasonable arguments based on antibody glycosylation which support the use of in vivo methods." (15)

Ascites is also subject to criticism on both technical and humane criteria. The major disadvantages of animal-based methods include: (1) association with severe cruelty and suffering to the animals; (2) ascitic fluids may be contaminated with rodent plasma proteins, immunoglobulins (reducing its immunoreactivity), infectious agents and bioreactive cytokines; (3) the need for extensive animal facilities, associated support services with each of the individual animals requiring daily monitoring 7 days a week; (4) some hybridomas (for example, human) are difficult to grow in rodents; (5) rodents only produce MAbs for a few days; (6) from 60 to 80 percent of mice may not produce ascites due to premature death, development of solid tumors, or failure to establish in vivo hybridoma growth (14); and (7) individual batches of ascites may vary significantly in quality and quantity.


Figure 1. Mouse showing swollen abdomen typical of ascites.

The most compelling arguments against in vivo production of monoclonal antibodies are based on the suffering associated with the induction of ascites in the animals. It is known from human clinical experience that the growth of abdominal tumors is very painful (fig. 1). Further, ascites fluid accumulation in human patients is associated with abdominal distension, anorexia, nausea, vomiting, respiratory distress, edema, decreased mobility and fatigue. Such individuals are treated for discomfort and pain while never being allowed to progress to advanced stages routinely seen during ascites production in animals.

Animals used for ascites accumulation of MAbs frequently exhibit a spectrum of clinical symptoms including: (1) roughened haircoat, hunched posture, abdominal distention, anorexia, cachexia, anemia; (2) decreased activity and body mass, dehydration, shrunken eyes; (3) difficulty walking, respiratory distress due to an elevated diaphragm; (4) circulatory shock due to excessive fluid removal; (5) decreased venous, arterial and renal blood flow; (6) classical peritonitis; (7) immunosuppression associated with adjuvant use; and (8) up to 20 percent mortality after removal of ascitic fluid. It is not uncommon for fluid to be withdrawn in amounts greater than the entire blood volume of the animal. These symptoms become increasingly severe the longer the animals are allowed to survive (1, 9).

Pathological changes associated with ascites production of MAbs are known for each step in the process. Use of adjuvants produces mild to severe peritonitis and inflammation. Fluid removal may cause hemorrhage, edema, and death. As expected, growth of the ascitic tumors creates a variety of responses including: (1) adhesions of the abdominal wall, bladder, diaphragm, kidneys, liver, seminal vesicles, testicles and ureters; (2) linear lesions in diaphragm muscles; (3) enlarged thoracic lymph nodes and lymphatic obstruction; (4) tumors with extensive hemorrhagic and necrotic areas; (5) disseminated tumors in mesenteric, lumbar, kidney, and testicular regions; (6) centro-lobular liver necrosis; and (7) solid tumors throughout the abdomen. (1, 9)

The significance of the list of abnormalities associated with ascites production is further emphasized by observations that the animals may experience severe pathologic changes in their abdomens and chests, but appear to be clinically normal. By themselves, frequent abdominal injections are known to be a major stressful factor. Further, although abdominal distention is the most obvious consequence of ascites production, severe pain from infiltrated growth of tumors and peritonitis is a significantly more serious problem for the animals.

From all of the above, it is apparent that animals used for ascites production of monoclonal antibodies are routinely subjected to chronic pain and distress. Use of adjuvants further complicates this situation by injuring the animals before the process begins.

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