BACKGROUND

The Guide for the Care and Use of Laboratory Animals and the PHS Policy on the Humane Care of Laboratory Animals requires that in vitro methods for antibody production be considered prior to the use of in vivo methods. The use of adjuvants for antibody production may promote robust immunity, however, the investigator needs to evaluate the effect of associated local and/or systemic pain and distress of the animal with the scientific benefit that may be gained. The use of inflammatory agents, particularly Complete Freund's Adjuvant (CFA), can result in severe side effects, including granuloma formation, tissue necrosis and sloughing, abscesses, and fever.^1-3 Alternatives to CFA should be used whenever possible and the use of CFA must be scientifically justified in the IACUC protocol.

Less problematic alternatives to Freund's adjuvant are available and should be considered. ^4-5 RIBI Adjuvant System®, Specol®, TiterMax®, Montanide IAS50, and Montanide ISA70 are commonly used as appropriate alternatives. Noninflammatory adsorptive adjuvants such as alum and aluminum hydroxide gel may also be considered.

A commonly used method for in-vivo monoclonal antibody production is the mouse ascites model. This method involves injecting a priming agent followed by hybridoma cells IP in mice. This generates ascites fluid, containing monoclonal antibodies, which is harvested from the mouse. When the ascites method is used for producing monoclonal antibodies, scientific justification is required and every reasonable effort should be made to minimize pain or distress, including frequent observation of the animal, limiting the number of survival taps, and prompt euthanasia if signs of distress appear.^6-7 

When utilizing adjuvants or using in-vivo methods to produce antibodies it is important to minimize the potential pain and distress associated with these procedures. The use of an analgesic is required unless robust scientific justification is provided and approved in the protocol.

IACUC Policy and Procedure

Adjuvant Use

1. When possible adjuvants other than CFA should be used. These adjuvants will produce a less intense inflammatory response (e.g. Aluminum compounds, squalene-in-water emulsions, monophosphoryl lipid A, RIBI adjuvants).
2. If CFA is required, it must be scientifically justified in the protocol. The protocol must state route of administration, number of injections, volume to be administered, concentration of CFA to be used, source of CFA, preparation of antigen-adjuvant emulsion (sterility, vehicle, pH), preparation of injection site, and monitoring of the animal.
3. CFA is considered to a chemical hazard and appropriate areas of protocol must be completed.
4. Recommendations when working with CFA:
   1. CFA should only be administered once. Incomplete Freund's adjuvant should be used for successive immunizations.
   2. Sterile technique must be used when preparing the antigen-adjuvant emulsions.
   3. Aseptic preparation of injection site.
   4. Adhering to recommended routes and volumes according to species (Table 1).
   5. Use of smallest possible volume for injections.
   6. Concentrations of CFA should be <0.1 mg/ml when possible.

Table 1. Recommended Volume (ml) of CFA-antigen Emulsion per Site and Route of Administration (NIH Guidelines)

*Strong scientific justification required

In-vivo monoclonal antibody production

1. Scientific justification is necessary for this procedure. The protocol must state reason that in-vivo method is required and in-vitro methods cannot be used, description of monitoring procedure, endpoints, number of collections, priming agent used (volume/route), hybridoma cells used (number of cells administered).
2. Administration of a priming agent is required for this model. Commonly used agents include Pristane. Incomplete Freund's Adjuvant (IFA) has also been shown to be an effective priming agent.
   1. Priming agents may induce discomfort so the lowest dose possible must be used.
   2. Recommendations: Pristane: 0.1-0.2 ml
3. Recommend waiting two (2) weeks between priming and hybridoma administration.
4. Administering a higher number of hybridoma cells is associated with greater morbidity.
5. Animals MUST be monitored daily:
   1. Baseline weight must be obtained and recorded.
   2. The animals must be weighed daily after hybridoma cell injections. The animals cannot gain more the 20% of starting weight.
   3. Animals must be observed closely for signs of pain or discomfort (hunched posture, rough haircoat, difficulty ambulating).
   4. Animal must be monitored for respiratory problems and signs of shock (pale eyes, ears, muzzle, and lethargy).
   5. If signs above are noted, animals must be euthanized prior to collection of fluids.
6. Fluid collection
   1. Ascites pressure must be relieved prior to abdominal distension causing discomfort.
   2. The peritoneocentesis can be performed under manual restraint or anesthesia.
   3. Aseptic technique must be followed.
   4. Maximum number of taps is three, with third being non-recovery.
   5. Animals should be monitored frequently over several hours following fluid collection to observe for signs of shock due to fluid withdrawal. 
      1. Preemptive fluid replacement with 1-2ml warm sterile saline SQ before the tap or IP immediately following the tap may alleviate some complications.
      2. If signs of shock are observed, animals must be culled.

REFERENCES

1. Jackson LR, Fox JG. 1995. Institutional policies and guidelines on adjuvants and antibody production. ILAR Journal 37(3):141-150.
2. Stills HF. 2005. Adjuvants and antibody production: dispelling the myths associated with Freund's complete and other adjuvants. ILAR Journal 46(3):280-293.
3. Toth LA, Dunlap AW, Olson GA, Hessler JR. 1989. An evaluation of distress following intraperitoneal immunization with Freund's adjuvant in mice. Lab Anim Sci 39:122-126.
4. Lipman, N.S., Trudel, L.J., Murphy, J.C., and Sahali, Y. 1992. Comparison of immune response potentiation and in vivo inflammatory effects of Freund's and RIBI adjuvants in mice. Lab Animal Sci 42: 193-97.
5. Leenars, M., Koedam, M.A., Hendriksen, C.F.M., and Claassen, E. 1998. Immune responses and side effects of five different oil-based adjuvants in mice. Vet Immunol Immunopath 61: 291-304.
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. Peterson, NC. 2000. Behavioral, clinical, and physiological analysis of mice used for ascites in monoclonal antibody production. Comp Med 50:516-526.

Approved: June 2018

Revision Approved: 7/2022