Each investigator is specifically responsible for ensuring that all those working with the animals are properly trained to perform the procedures and are aware of the contents of these guidelines and the ARC approved animal care protocol.

I.   JUSTIFICATION FOR THE ASCITES METHOD

The National Research Council (NRC) Committee on Methods of Producing Monoclonal Antibodies (http://grants.nih.gov/grants/policy/antibodies.pdf) states: 

• "It is incumbent on the scientist to consider first the use of in vitro methods for the production of mAb.  When hybridomas fail to grow or fail to achieve a product consistent with scientific goals, the investigator is obliged to show that a good-faith effort was made to adapt the hybridoma to in vitro growth conditions before using the mouse ascites method." 

• "In vitro methods for producing mAb are appropriate in numerous situations, and it is the responsibility of the researcher to produce scientific justification for using the mouse ascites method."   

Although the ARC may approve the use of the mouse ascites for the production of mAbs, a proposal to use this method must contain sufficient information for the Committee to evaluate whether or not there is adequate justification.  Ease of purification, higher antibody yield, and lower cost are not acceptable reasons to use the ascites method unless carefully and properly justified. 

1.   Failure of In Vitro Methods to Produce the Monoclonal Antibody

a.       Examples:

• Some cell lines do not adapt well to tissue culture conditions.

• Rat cell lines usually do not efficiently generate mAb in rats and adapt poorly to tissue-culture conditions but do produce mAb in immunocompromised mice.

b.      Justification must address the following:

• Has in vitro monoclonal antibody production been attempted by the investigator and/or an outside source?  If so, is supporting data available to demonstrate that the in vitro method was unsuccessful in producing the monoclonal antibody using several different culture conditions (cell lines and serum-free medium)?
• Has the investigator considered an NIH-assured commercial source or other core hybridoma facility for ascites production?  

2.   Problems with Antibody Production/Yield Using In Vitro Methods

a.       Examples: 

• Inability of some cell lines that do adapt to tissue-culture conditions to maintain adequate production of mAb poses a serious problem.

• In applications where several different mouse mAb at high concentrations are required for injection into mice, the in vitro method can be inefficient.

b.      Justification must address the following:

• If low antibody production is obtained using the in vitro method in an investigator's laboratory, is the mAb available commercially?
• Is supporting data available to demonstrate that insufficient antibody production was obtained by the investigator and/or an outside source using the in vitro method?
• What technical reasons resulted in difficulties obtaining the required level of antibody yield using the in vitro method? Specify culture conditions, medium, culture systems, cell lines, and indicate whether cell lines were tested for mycoplasma.

a.       Examples:

• Downstream purification or concentration from in vitro systems can lead to protein denaturation and decreased antibody activity.

• Tissue-culture methods can yield mAb that do not reflect the normal modification of proteins with sugars, and this abnormality might influence binding capacity and other critical biologic functions of mAb. 

b.      Justification must address the following:

• What technical reasons specific to the study resulted in difficulty obtaining the required antibody using the in vitro method?

a.       Examples:

• Prohibitive cost of purchasing mAbs from outside sources using the in vivo or in vitro method.

• Prohibitive cost of in vitro mAb production in an investigator's laboratory.

b.      Justification must address the following:

• If cost is used as a justification, the source of this information and a cost estimation for producing the total amount of antibody required must be included.  When providing this information, please include:

  • your estimated cost for the in vivo and in vitro methods. In estimating your cost, please include the cost of maintaining and housing animals, labor associated with animal care, and purification of the mAb; and

  • vendor's cost for both the in vivo and in vitro methods. 

5.   In Vivo Monoclonal Antibody Production at Other Institutions

Please provide the following information:

• Name of organization producing the antibodies

• Organization's Animal Welfare Assurance Number on file with the NIH Office of Laboratory Animal Welfare (OLAW).  A list of institutions holding Assurances with OLAW is available at http://grants.nih.gov/grants/olaw/olaw.htm#assur.

• Organization's USDA Registration Number.  A list of USDA-registered facilities is available at http://www.aphis.usda.gov/animal_welfare/efoia/index.shtml.  

• Organization's accreditation status with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.  A directory of AAALAC-accredited institutions is available at http://www.aaalac.org/accreditedorgs/index.cfm.

II.  RESOURCES

A listing of commercial sources of in vitro production methods and commercial sources of mAbs is available at http://www.nal.usda.gov/awic/pubs/antibody/company.htm.

III. ALTERNATIVES

Federal regulations require investigators to consider alternatives to procedures that may cause more than momentary or slight pain or distress to animals.  As ascites production in mice may cause more than momentary or slight pain or distress to animals, scientists are obligated to consider alternatives to the ascites method. Failure to use in vitro methods in favor of the in vivo method must be scientifically justified, and the ARC will not approve the use of the mouse ascites method in the absence of such a justification.  At a minimum, federal regulations require that investigators provide a written narrative with the following information: date(s) of the most recent literature search, years covered by the search, keywords used to search for alternatives, and the databases or sources consulted to support the conclusion that no alternatives are applicable (e.g., Medline Plus, BIOSIS, PsycINFO, Current Contents, etc.).

Several resources are available to assist investigators in the search for alternatives.  Please visit the Office of Animal Research Oversight website at http://oaro.research.ucla.edu/alternatives for more information.

IV.  GUIDELINES FOR THE ASCITES METHOD

1.   Pain Categorization

Provided that animals are monitored appropriately and that ascites fluid is tapped before the animals are expected to experience unalleviated pain or discomfort as a result of the accumulation of fluid in the peritoneal cavity, the ARC has determined that animals used for ascites production may be listed under Pain Category D (pain/distress relieved by use of appropriate anesthetics, analgesics, tranquilizers, or by euthanasia).  However, if the experiments are expected to produce significant inflammation or otherwise result in unrelieved pain, Pain Category E (pain/distress cannot be relieved by use of anesthetics, analgesics, or tranquilizers, as the use of these agents would interfere with the experimental design) may be appropriate.

2.   Priming

Pristane, the most commonly used priming agent for ascites production, is believed to act by inducing a granulomatous reaction and by interfering with peritoneal fluid drainage.  A dose of 0.5 ml i.p. may cause noticeable distress, which is not seen with the equally efficacious dose of 0.1 to 0.2 ml.  Therefore, pristane-priming must be performed with doses no greater than 0.2 ml per injection unless scientifically justified in the animal use protocol.

Scientific justification must be provided in the animal use protocol for the use of priming agents other than pristane. 

3.      Inoculation

Tumor cell lines should be tested for murine virus.  Untested mice, or those contaminated with murine viruses, must be isolated.  Hybridoma cell suspensions in 0.5 ml of media is recommended for increased mAb production.

After inoculation with ascites-producing tumor lines, mice must be observed at least twice daily (including weekends and holidays) to monitor the degree of abdominal distention and for signs of clinical abnormalities and distress (e.g., hunched posture, rough haircoat, dehydration, or difficulty in ambulation). The animals must be weighed at least every other day beginning seven days after hybridoma injection. 

Animals having difficulty reaching their food or water should have food pellets and Napa Nectar (or other water source) placed inside their cage.

4.      Harvesting Ascites Fluid

Accumulation of fluid in the peritoneal cavity causes abdominal distention, discomfort, and in extreme cases may even cause respiratory distress.  Ascites fluid must be removed before abdominal distention is such as to cause discomfort, tachypnea, or interference with normal activity.  Any animal with a grossly distended abdomen, one in which the skin is drawn tight, must be tapped and/or euthanized.  Animals must be tapped before ascites fluid volumes exceed 20% of the animal's baseline body weight prior to hybridoma cell inoculation.

Ascites fluid may be collected with a sterile 18-22 gauge hypodermic needle (glass syringes have been found to be more effective for this procedure than plastic).  Passive flow from the needle usually works best.  The collection procedure should be performed using aseptic techniques, including clipping or shaving the fur from the injection area, preparation of the site with 3 alternating washes of both betadine and 70% ethanol, and using a new, sterile hypodermic needle for each mouse.  To minimize discomfort, the needle should be inserted lateral to the bladder and parallel to the body wall.  Anesthesia is not normally required if those performing the collection are experienced.  The animals should be anesthetized if new personnel are being trained.

Animals must be observed continuously by trained personnel at least 30 minutes immediately following abdominal paracentesis for signs of hypovolemic shock and distress.  If an animal appears hunched or lethargic, an equal volume of warm saline should be administered subcutaneously. 

At a maximum, animals may be tapped twice and allowed to recover.  The third tap, if one occurs, must be conducted following euthanasia.

5.      Euthanasia

Animals bearing ascites tumors must not be maintained past a point where they are in good health.  Animals must be euthanized promptly if they display severe signs of pain or distress or exhibit severe or persistent clinical abnormalities (ruffled coat, hunched posture, anorexia, dehydration, pallor, weight loss, inactivity, difficulty in ambulation, tachypnea, or dyspnea).  Any animal in the moribund condition must be euthanized.

REFERENCES

Amyx, H.L. 1987. Control of animal pain and distress in antibody production and infectious disease studies. JAVMA 191(10):1287-1289.

Brodeur, B.R., Tsang, P. and Larose, Y. 1984. Parameters affecting ascites tumor formation in mice and monoclonal antibody production. J. Immunol. Methods 86:239-241.

Colwell, D.E., Michalek, S.M. and McGhee, J.R. 1986. Method for generating a high frequency of hybridomas producing monoclonal IgA antibodies. Methods Enzymol. 121:42-51.

Jackson, L.R. and Fox, J.G. 1995. Institutional policies and guidelines on adjuvants and antibody production. ILAR Journal 37(3):141-152.

Jackson, L.R., Trudel, L.J., Fox, J.G., et al. Monoclonal antibody production in murine ascites II. Production characteristics. Laboratory Animal Science 49(1):81-86.

McGuill, M.W. and Rowan, A.N. 1989. Refinement of monoclonal antibody production and animal well-being. ILAR News 31(1):7-10.

Monoclonal Antibody Production Guidelines, Animal Care and Use Administrative Committee (AUCAAC), University of California, Davis, 8/12/99.

Production of Monoclonal Antibodies Using Mouse Ascites Method. OPRR Reports, Number 98-01, Animal Welfare, National Institutes of Health, November 17, 1999.

Ward, P.A., Adams, J., Faustman, D., et al. Monoclonal Antibody Production: A Report of the Committee on Methods of Producing Monoclonal Antibodies. Institute for Laboratory Animal Research, National Research Council, National Academy Press, Washington D.C. 1999.

Guidelines for Ascites Production in Mice.  http://oacu.od.nih.gov/documents/Ascites.pdf, last revision 9/12/07.

Approved 2/14/00

Revised 11/13/00, 3/22/04, 4/26/10