Antibody Production

A. Monoclonal Antibody Production

This Standard Procedure does not cover in vivo production of monoclonal antibodies via IP hybridoma cell ascites production. If this method of in vivo monoclonal antibody production is required, it must be fully described and justified in the approved IACUC protocol. Institutional Animal Care Use Committees are expected to critically evaluate protocols that propose using the mouse ascites method, and only allow in vivo production based on strong scientific justification.

IACUC Alternatives to Ascites Production of Monoclonal Antibodies:
Both the OLAW and the USDA encourage the use of alternative methods to produce monoclonal antibodies in vitro without compromising the aims of the study. 

B. Polyclonal Antibody Production

The IACUC protocol must describe these aspects of the antibody production procedure:

1. Antigen: The immunizing antigen needs to present no known risk of pathogenicity or toxicity to the host animal. If the use of a known toxic or pathogenic antigen is necessary, its use must be justified.

2. Adjuvant: Adjuvants may enhance a response, but many cause moderate to severe inflammation at the site of administration. Most of the undesirable sideeffects of polyclonal antibody (pAb) production are caused by the adjuvant6. For all adjuvants, adverse effects can be reduced by using small inoculum amounts and a recommended administration method (see #3 below). Follow the manufacturer’s guidelines for all adjuvant use.  

   Use of Complete Freud’s Adjuvant (CFA) should be avoided as it may cause severe inflammation or necrosis. Other adjuvants (Table 1, below) should be considered before using CFA. If CFA cannot be avoided, justification must be provided in the protocol, and CFA should only be used for first (priming) dose. 

3. Immunization (of Immunogen/Adjuvant): 

   Adjuvants other than CFA, see Table 1: Subcutaneous (SC) or intraperitoneal (IP) administration is preferred in rodents. Injection volume in both mice and rats should not exceed 0.1 ml SC; 0.2 ml IP. IP is permissible only in rodents and may only be administered as a single injection.

   CFA: The subcutaneous route is preferred in rodents, with injection volume not exceeding 0.1 ml SC in mice and rats. IP injection of CFA in mice and rats has been documented to cause severe peritonitis9,12 and is generally not recommended for polyclonal antibody production.11

   All adjuvants: The use of multiple subcutaneous injection sites containing small volumes (e.g., 0.05 mL per site) distributes the immunogen/adjuvant over a larger surface area for the immune system to process the antigen, resulting in higher titers while also reducing the incidence of a severe local inflammatory response and abscessation. Multiple injection sites should be sufficiently separated to prevent the potential coalescing of the inflammatory lesions, which may result in disruption of blood supply to the area and subsequent formation of draining abscesses or tissue sloughing. Injections at multiple sites should not be more than 0.05 ml per site for mice and rats. Other routes and/or sites, such as footpad (ID or SC injection into the hindfoot sole surface aka intraplantar injection), must be described and justified in the protocol. 

4. Blood Collection: Maximum withdrawal should not exceed the volumes defined in the UCSF Guidelines for blood collection in mice and rats.
5. Titer Sampling Routes: Titer sampling should be performed using the submandibular vein in mice and a peripheral vessel for rats and other species. 
6. Vasodilation: Radiant heat or warm water is recommended.
7. Monitoring: Investigators assume responsibility for ensuring their animals' health through adequate monitoring. If complications arise, you must consult veterinary staff, or alternatively euthanize animals.

Agents: This procedure may require anesthesia. All agents administered to animals should be listed in the "Agents" section of RIO.

Potential adverse effects to be considered: shock, peritonitis, inappetence, swelling, abscesses or ulceration at the injection sites

Table 1. Adjuvants other than CFA may include, but are not limited to:

1. aluminum compounds (e.g., Alum)
2. squalene-in-water emulsions (MF59 and AS03)
3. monophosphoryl lipid A (MPL)
4. Ribi adjuvants, combined with alum (AS04)
5. adjuvants in pre-clinical development (e.g., Montanides)
6. polymeric microparticles
7. saponins (e.g., Quil A QS-21, ISCOMS, ISCOMATRIX)
8. immunostimulatory nucleic acids (e.g., CpG oligodeoxynucleotides, poly IC:LC)
9. other toll-like receptor-agonists (e.g., flagellin, imidazoquinolines, small molecules)
10. cationic liposome formulations (CAF) combined with immunostimulators such as trehalose dibehenate (TDB) virus-likeparticles, nanoparticles, and oligonucleotide complexes
11. emulsions (e.g., TiterMax, Syntex Adjuvant Formulation (SAF), and Specol or Stimune)

References

1. Amyx, H.L., (1987), Control of pain & distress in antibody production & infectious disease studies. Journal of the American Veterinary Medical Association, Nov. 15, 1987, v. 191 (10), p. 1287-1289. ISSN: 0003-1488.
2. ARAC. Guidelines for the Use of Adjuvants in Research: Special Emphasis on Freund’s Adjuvant. NIH Intramural Guidelines.2019.
3. Broderson JR. A retrospective review of lesions associated with the use of Freund's adjuvant. Lab Anim Sci. 1989;39(5):400-5. 
4. Guidelines for the Production of Polyclonal and Monoclonal Antibodies in Rodents and Rabbit, Research Animal Resource Center, Memorial Sloan-Kettering Cancer Center, Weill Cornell Medical College, 2015
5. Guidelines on: Antibody Production, Canadian Council on Animal Care, 2002
6. Jennings VM. Review of Selected Adjuvants Used in Antibody Production. ILAR J. 1995;37(3):119-125. doi: 10.1093/ilar.37.3.119. PMID: 11528031.
7. Kolstad, JAALAS, 2012
8. Leenaars PP, Hendriksen CF, Koedam MA, Claassen I, Claassen E. 1995. Comparison of adjuvants for immune potentiating properties and side effects in mice. Vet Immunol Immunopathol 48:123–138.
9. Leenaars PP, Koedam M, Wester P, Baumans V, Claassen E, Hendriksen CF. Assessment of side effects induced by injection of different adjuvant–antigen combinations in rabbits and mice. Lab Anim Lab Anim. 1998 Oct;32(4):387-406. doi: 10.1258/002367798780599884. PMID: 9807752.
10. Leenaars, M., & Hendriksen, C.F. (2005). Critical steps in the production of polyclonal and monoclonal antibodies: Evaluation and recommendations. Institute for Laboratory Animal Research Journal, 46(3), 269-79.
11. Leenaars PPAM, Hendriksen CFM, de Leeuw WA, Carat F, Delahaut Ph, Fischer R, Halder M, Hanly WC, Hartinger J, Hau J, Lindblad EB, Nicklas W, Outschoorn IM, Stewart-Tull DES. 1999. The Production of Polyclonal Antibodies in Laboratory Animal. The Report and Recommendations of ECVAM Workshop 35. ATLA 27:79-10"
12. Stills HF Jr. Adjuvants and antibody production: dispelling the myths associated with Freund's complete and other adjuvants. ILAR J. 2005;46(3):280-93. doi: 10.1093/ilar.46.3.280. PMID: 15953835.
13. 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.
14. Toth, LA; and Olson, GA. 1990. Strategies for minimizing pain and distress in laboratory animals. Lab. Anim. 20(4):33-39.
15. UW, Office of Animal Welfare, “Use of Complete Freund’s Adjuvant (CFA) and Other Adjuvants in Research Animals.”

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