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IACUC Guideline IV
Use of Rodents in Ascites Production of Antibody

Background
Specific Guidelines
Available Help

Background:

The following guidelines for ascites production reflect the findings of the National Research Council of the National Academy of Sciences' report, Monoclonal Antibody Production, released in April 1999. This study was commissioned by the National Institutes of Health (NIH) in response to petitions by the American Anti-Vivisection Society and others. These petitions requested a ban on the use of ascites production.

Background: The overall finding of the 1999 National Academy of Sciences report was that while in vitro methods of monoclonal antibody production should be used whenever possible, there are legitimate circumstances for use of the ascites method. The primary consideration in these instances is the minimization of pain and distress to the rodents used. The aspects of the ascites protocol that need to be controlled so as to minimize pain and distress include priming, hybridoma implantation, and the harvest of ascites fluid.

Priming: Pristane (2,6,10,14 tetramethylpentadecane), and less often, incomplete Freund's adjuvant (IFA) are agents injected into the peritoneal cavity prior to hybridoma implantation to facilitate the formation of ascites. Variables in the priming step are choice of agent, dose, and timing. IFA has been used as an alternative to pristane1, 2 and it has been suggested that its adverse effects are less1. The use of IFA has been relatively uncommon, however, therefore its general applicability is unknown. Several studies have now been published3, 4 that indicate that doses of pristane of 0.25 ml or lower will produce an equal volume of ascites to the more usual 0.5 ml dose, with milder adverse effects5. While a recent study on the effects of pristane speculated that the peritonitis induced by this agent could cause significant distress to the animal, the authors were unable to document clinical abnormalities following injection of 0.5 ml6.

Implantation: The number of cells implanted into the peritoneal cavity can vary, but there is an inverse relationship between dose and the speed of induction of ascites fluid, as well as expected lifespan7. The recommended dose is 5 x 105 to 5 x 106 in a 1 ml volume7, 8.

Harvest: It is critical that animals with implanted hybridomas be monitored daily for signs of abdominal distension, as ascites fluid needs to be drained soon after accumulation to minimize mortality5. In a study by Jackson, et.al.6, groups of animals subjected to multiple ascites taps were found to have minimum survivals of 85% for the first two taps, dropping as low as 35% in some groups for the third tap. Clinical abnormalities consistent with circulatory shock were seen in some animals in this study. Many institutions have established policies limiting abdominal taps, usually to three, with the final tap being terminal.

Specific Guidelines:

SPECIFIC GUIDELINES - Selection of Animals:

  1. Only healthy, viral antibody-free (VAF) animals should be selected for ascites production to maximize the probability of producing high titer ascites fluid and to decrease the chance of producing ascites fluid contaminated with infectious organisms.
  2. Retired breeders are superior to young animals since the abdominal distention associated with ascites production is better tolerated and greater volumes of ascites fluid can be obtained.

Priming Agents:

  1. Pristane: The dose of pristane administered intraperitoneally should be limited to that which is found to induce ascites formation, and should not exceed 0.3 ml per mouse. A recommended starting dose is 0.2 ml. per mouse, and in some instances a dose of 0.1 ml may be sufficient. Pristane should be administered 1-3 weeks prior to hybridoma implantation.
  2. Incomplete Freund's Adjuvant (IFA): A dose of 0.25 - 0.50 ml may be injected intraperitoneally. Hybridoma implantation may be as soon as 24 hours following injection of IFA.

Hybridoma Implantation:

  1. Following the appropriate priming period, 5 to 10 x 106 viable hybridoma cells should be injected in a volume of 1 ml or less of a sterile, physiological saline or balanced salt solution.
  2. Only cell lines demonstrated to be clear of murine viruses should be used in conventional animal facilities. It is the investigator's responsibility to work with the Animal Resource Program (ARP) to ascertain the status of the tumor cell line they intend to use.
  3. Hybridoma lines obtained from outside the University should be MAP (Mouse Antibody Production) or PCR tested for pathogens before being injected. Commercial sources of cell lines, such as ATCC, are typically not MAP tested by the supplier. Details on MAP testing are available through ARP.

Animal Monitoring:

  1. All primed animals, and those implanted with hybridomas, should be observed daily for signs of distress or poor health. The observer must be capable of recognizing signs of distress and able to take appropriate actions when indicated. It is ultimately the investigator's responsibility to monitor the animals' health, and to ensure that animals with abdominal distension can continue to reach food and water.
  2. Animals that become moribund or exhibit significant distress (defined here as seriously ill- lethargic, ruffled coat, hunched posture, weight loss, dehydrated, hypothermic) should be euthanized by the Institutional Animal Care and Use Committee (IACUC) approved methods such as CO2 asphyxiation, cervical dislocation or anesthetic overdose.

Obtaining Ascites Fluid: Ascites fluid should be obtained as soon as abdominal distension is obvious, but before respiration becomes labored.

  1. Disinfect the abdominal surface with 70% alcohol.
  2. Tap the abdomen using a 1-inch, 18 to 22-gauge needle. Allow fluid to drain into a suitable sterile collection tube. This can best be accomplished by restraining the animal in one hand, positioned about 45 degrees from horizontal, while holding the collection tube in the other hand.
  3. Anesthesia or sedation can be used to minimize the pain and distress of the procedure. Halothane administered by inhalation is the anesthetic of choice. There is an increased risk of anesthetic death if the abdominal enlargement due to ascites is sufficient to compromise respiration.
  4. Ascites production is debilitating to the animal due to peritoneal inflammation, growth of the hybridoma and associated accumulation of fluid. The general condition of implanted animals should be assessed following each tap, observing especially for loss of weight. Animals that have lost excessive weight (greater than 25% of initial) should be euthanized.
  5. Two abdominal taps will usually prove sufficient, as, in many instances, little additional fluid will be produced after the second tap. A maximum of three abdominal taps may be collected if the animal shows no signs of inordinate distress or debilitation from the procedure.
  6. At the conclusion of the final tap, animals should be euthanized using an IACUC approved method.

Available Help:

HELP IS AVAILABLE - The veterinary and technical staffs of ARP are available for consultation on these procedures. They may be reached at 814-865-1495.

References:

  1. Division of Comparative Medicine, MIT. (1990). Guidelines for the Production of Polyclonal and Monoclonal Antibodies in Rodents and Rabbits. Appendix 1, pg. 5.
  2. Gillette, R.W. (1987). Alternatives to Pristane Priming for Ascitic Fluid and Monoclonal Antibody Production. J. Immunol. Meth. 99, 21-23.
  3. Hoogenraad, N.J. and Wraight, C.J. (1986). The Effects of Pristane on Ascites Tumor Formation and Monoclonal Antibody Production. Meth. Enzymol. 121, 375-85.
  4. Montgomery, C.A. (1990). Oncologic and Toxicologic Research: Alleviation and Control of Pain and Distress in Laboratory Animals. Cancer Bulletin 42(4), 230-37.
  5. Brodeur, B.R., Tsang, P., and Larose, Y. (1984). Parameters Affecting Ascites Tumour Formation in Mice and Monoclonal Antibody Production. J. Immunol. Meth. 71, 265-72.
  6. Jackson, L.R., Trudel, L.J., Fox, J.G., and Lipman, N.S. (1999). Monoclonal Antibody Production in Murine Ascites. I. Clinical and Pathologic Features. Lab. Animal Sci. 49, 70-80.
  7. Rowsell, H.C. (1989). Guidelines on Immunologic Procedures. SCAW Newsletter 11, 2-7.
  8. Harlow, E. and Lane, D. (1988). Growing Hybridomas. In Antibodies, a Laboratory manual. Cold Springs Harbor Laboratory. 245-82.

Last Revised by the IACUC on 12/13/2001
Last Approved by the IACUC on 12/08/2008