The use of an anti-CD40 agonist monoclonal antibody during immunizations enhances hybridoma generation.

ABSTRACT Herein we describe the use of an agonistic anti-murine CD40 MAb as a B cell proliferative agent to enhance the generation of monoclonal antibodies (MAbs) in Balb/c mice. While hybridoma technology has been validated repeatedly over the decades, little work has been described to improve upon the overall numbers of in vivo B cells and specific antibodies obtained from a fusion. To begin to address this situation, strategies to boost B lymphocyte yields for hybridoma production were employed. Anti-CD40 agonist antibodies have been reported to activate and amplify human resting B lymphocytes in vitro, resulting in increased cell numbers available for the generation of human hybridomas or B cell clones. An agonistic anti-murine CD40 MAb was administered to immunized mice 3 days prior to splenic harvest, and B lymphocyte yields were found to be approximately 2-fold higher in treated animals when compared to untreated animals. Moreover, the resulting hybridoma fusions using lymphocytes from treated animals yielded 5- to 10-fold more antigen reactive hybrids when compared to untreated animals. This novel addition to conventional approaches utilizes the proliferative effects of agonistic anti-CD40 MAbs to markedly enhance monoclonal antibody generation.