NEGATIVE EFFECT OF MULTIPLE ANTIGEN INJECTIONS ON THE YIELD OF MURINE MONOCLONAL-ANTIBODIES OBTAINED BY HYBRIDOMA TECHNOLOGY

One of the critical steps in the preparation of monoclonal antibodies is the obtainment, by in vivo immunization and boost, of the maximal number of antigen-activated B lymphocytes. In hybridoma laboratories, the common procedure is to immunize a group of mice with several antigen injections and use the mouse showing the highest serum antibody titre for the fusion experiment. The observation that the use of mice hyperimmunized with human red blood cells failed to yield a high number of monoclonal antibodies, led us to study the effect of multiple antigen injection prior to the fusion experiment. The results obtained showed that the maximal yields of monoclonal antibodies were obtained using mice that had received only one or two antigen injections while the mice immunized with three antigen injections consistently yielded at least a three-fold reduction in the number of monoclonal antibodies. The negative effect could not be reversed by prolonged resting of the animals and suggests the induction of a tolerance/suppression state which can prevent the final activation step. These results point out to the importance of avoiding the hyperimmunization of mice for the preparation of a high number of monoclonal antibodies by standard hybridoma technology.