When the mouse begins to produce antibodies to the antigen, its spleen is removed. The new fused cell line, which does produce antibodies, is grown briefly in culture and then re-injected into another mouse's peritoneum. Finally, the ascites fluid which contains monoclonal antibodies is harvested from the mouse. The purity of the antigen, the mouse strain which is used, the age and sex of the mouse, the tolerance of the individual mouse, and many other factors may affect the outcome of Ab-producing procedures. Because so many variables are present, one general way to maximize the animal's immune response is to use adjuvants.
Adjuvants hold the antigen at the site of immunization and release it over a long period of time.
Freund's Complete Adjuvant, which is more widely used than any other adjuvant, includes killed mycobacteria which serve to increase inflammation upon injection, and thereby enhance the mouse's immune response. This inflammation often results in painful lesions at the injection site. This system is widely used because it can process large numbers of samples in a short time and can handle complex antigens, such as bacteria. In this assay, the MAb is an antigen which has been anchored to a surface. This antibody then can be detected by a polyclonal antibody preparation which binds the monoclonal antibody.