Office of Research Integrity

Production of Monoclonal Antibodies

*MUSC has adopted the Canadian Council on Animal Care (CCAC) Guidelines for monoclonal antibody production in the mouse with slight modifications based on current literature.2


The most important aspect of hybridoma production is the utilization of skilled, competent, technical staff experienced in the handling of the species being used and in the conduct of the technique. They must be knowledgeable and capable of recognizing signs of distress in all injected animals, and be responsible for taking action when necessary.

Induction of Ascites fluid in animals:

Pristane or other recognized priming agent(s) (excluding FCA) may be used.

Ascites may be collected only for as long as the animal is not experiencing pain or distress, is in good body condition, and does not show signs of debilitation, dehydration or other complications from the procedure. Upon recognition of loss of condition, pain or distress, the animal must be euthanized according to a method approved by the CCAC.

Example of acceptable standard operating procedure for the production of monoclonal antibodies in the mouse*

  1. Primary Immunization
    • The mouse is immunized with antigen emulsified in Freund's complete adjuvant (CFA):
      • mix 0.1 ml of antigen with CFA (equal parts)
        0.1 ml:0.1 ml
    • Inject 0.05 ml IP into the mouse
    • Harvest the spleen, following euthanasia, at the appropriate time.
  2. Pristane Priming
    • Inject 0.1 – 0.2 ml of pristine IP 14 days before the injection of the hybridoma. 1
  3. Injection of the Hybridoma
    • Inject approximately 320 x 10 4 cells per mouse, IP
    • Mice will begin to produce ascites fluid approximately 4 to 8 days after injection and will continue to do so for approximately 6 days
    • The ascites fluid is collected by tapping the abdomen of the mouse using a 22 g needle and allowing the fluid to drain into a test tube through the hub of the needle. (A small amount of heparin may be added to the collection tube to prevent clotting – according to investigator instructions.)
    • Ascites fluid may be collected for as long as the mouse is not experiencing pain or distress, is in good body condition, and does not show signs of debilitation or complication from the procedure.
    • Upon the recognition of loss of condition, pain or distress, the mouse must be euthanized.
  4. Animal Model
    • often the male BALB/c mouse of 43 to 78 days of age

1 Olfert, E.D., B.M. Cross and A.A. McWilliam, eds. Guide to the Care and Use of Experimental Animals . Canadian Council on Animal Care. Vol 1 (1993) 173-174.

2 McGuill, M.W. and A.N. Rowan. “Refinement of Monoclonal Antibody Production and Animal Well-Being.” ILAR News , 31, No. 1 (1989) 7-10.

- Courtesy University of Ottawa