The present status of established policies and procedures on adjuvant use and monoclonal antibody production is under review by both the scientific community and regulatory agencies, specifically, the Office of Protection from Research Risks (OPRR) of the NIH. Recent communications from the OPRR to grantee institutions strongly urge Institutional Animal Care and Use Committees to fulfill their obligation of assuring the welfare and humane care and use of animals by exploring and adopting procedural refinements and to utilize in vitro alternatives to classic mouse ascites production for preparation of large quantities of monoclonal antibodies. Certain alternative study centers, animal welfare groups, regulatory agencies and scientific societies now consider monoclonal antibody production using the mouse ascites method to be outdated and unjustified on humane grounds since there is evidence that such methodology causes discomfort distress or pain and cost effective, satisfactory alternative in vitro methodologies now exist for production of significant quantities of pure monoclonal antibodies. A timely review of appropriate procedures and policies concerning monoclonal antibody induction and production appears warranted. Selection and use of any methodology, whether animals are involved or not, should be based upon sound scientific rationale. A great place to lok for detailed assessments are the works of Jackson, L.R. and Fox, J.G., titled Institutional Guidelines and Policies on Adjuvants and Antibody Production. (ILAR Journal 37(3):141-152, 1995).

The polices of the Duke University animal care program continue to allow monoclonal antibody production using the mouse ascites method with certain restrictions as noted below.

Selection of Animals: Although many different mouse strains can be used for immunization, there are strain differences in responses. Most hybridomas have origins in Balb/c plasmacytomas and spleen cells and hybridoma recipient mice must also be Balb/c strain or immunodeficient animals have to be used. Whereas, female retired breeder mice are preferred because of availability, large size and tendency not to fight, other researchers prefer the use of Balb/c derived crossbred F1 hybrids because of reported greater ascites volumes and antibody production.

Immunization Procedures: Immunization schedules vary depending upon the nature of the antigen. Intravenous (tail vein) inoculations (0.1-0.5ml) are routinely used for particulate antigens and subcutaneous (0.05-0.2ml) and intra peritoneal (up to 0.5ml) routes are generally used for antigen/adjuvant mixtures. Intradermal injections should be limited to 0.05ml. Intramuscular injections are discouraged. Complete Freund’s adjuvant (CFA)/antigen mixtures should be limited to primary immunizations and Incomplete Freund’s Adjuvant (IFA) should be used in subsequent booster inoculations at 14 to 28 days after primary immunization. The antibody titers are generally determined in blood samples taken at 10 to 24 days after the boost and, when peak titers are reached, animals are rested for 3-4 weeks. Mice are then given a final intravenous aqueous antigen boost and euthanatized by cervical dislocation or CO2 narcosis 3 days later. Animals failing to achieve high antibody titers may be boosted multiple times.

Successful in vitro immunization schemes have been reported with noted advantages such as shortened immunization time to days instead of weeks, only very small amounts of antigen are required and a broader library of antibodies can be produced because of lack of potential compromising in vivo phenomena such as suppression or tolerance. Regardless of the method or antigen mixture used, all antigen preparations should be prepared aseptically using sterile vehicles and final mixtures should be cultured for presence of contaminating bacteria.

Priming Agents: Priming agents to prevent solid tumor production and promote ascites production are generally used IP prior to IP inoculation of hybridoma cells. Pristine (2,6,10,14 tetramethylpentadecane) (0.2-0.5ml) is the most frequently used ascitogenic priming agent although CFA, IFA, proteose-peptone, thioglycollate, corn oil and mineral oil have also been successfully utilized.

Inoculation of Hybridoma Cells: The optimal time interval between pristine priming and aseptic, IP, hybridoma inoculation is between 1 and 4 weeks which allows for establishment of appropriate levels of peritoneal inflammatory cells. Although the optimal number of hybridoma cells recommended for inoculation may be dependant upon the biologic behavior of the cell line, such cell dosages resulting in greatest ascites volume and antibody concentrations generally range between 106 and 107cells. Hybridomas should be tested for the presence of adventitious viral and mycoplasma agents prior to inoculation into mice in order to prevent potential transmission of murine infectious agents into animal facility experimental colonies.

Clinical Observations: Every other day clinical observations of inoculated animals is sufficient for the first few days. However, once ascites fluid accumulation and peritoneal cavity distention is noted, daily observations, including weekends and holidays, of animals are required. Animals should be observed for obvious distress or discomfort, rapid or labored breathing, palor, hunched posture, inactivity, dehydration, weight loss, roughened hair coat and ambulation difficulty. Mice showing signs of distress or moderate clinical effects of abdominal distention should undergo euthanasia and paracentesis to retrieve ascites fluid. The policy of the Duke IACUC does not allow mice to progress to a state of full peritoneal filling with abdominal rigidity. If animals exhibit severe clinical signs or become moribund, they should be euthanatized in a timely manner.

Abdominal Paracentesis: The policy of the Duke IACUC does not allow for repeated tapping of ascitic fluid from hybridoma bearing mice. Therefore, paracentesis should be performed only on euthanatized animals. Fluids should be retrieved by abdominal insertion of a long stem Pasteur pipette or large (16-18) gauge needle. If abdominal distention cannot be relieved by paracentesis, peritoneal solid tumor growth should be suspected.