An efficient immunization protocol for production of monoclonal antibodies against soluble human insulin

A new immunization protocol has been developed to obtain specific monoclonal antibodies (mAb) directed against the soluble form of human insulin. Various protocols differing on the basis of the nature of immunogen, the number of injections and the route of administration of the antigen were compared. Mice with the highest anti-insulin titers were selected for cell fusion. The results showed that the immunization protocol involving 2 injections of insulin followed by a boost 2 months later mainly stimulated B lymphocytes secreting IgM mAb directed against immobilized insulin. Immunization with 2 injections of human proinsulin followed by 2 injections of a human insulin-bovine serum albumin conjugate and finally with a booster injection of this conjugate on each of the last 4 days preceding fusion was necessary to obtain a high percentage of hybridomas secreting specific IgG mAb able to recognize immobilized insulin (indirect ELISA) as well as iodinated insulin (liquid-phase radioimmunoassay).