HUMAN MONOCLONAL OR POLYCLONAL ANTIBODIES RECOGNIZE PREDOMINANTLY DISCONTINUOUS EPITOPES ON BEE VENOM PHOSPHOLIPASE A(2)

Background: Two hybridomas, which secrete human monoclonal antibodies of IgG(4) isotype specific for the main bee venom antigen/allergen pho spholipase A(2) were generated. The antigenic determinants recognized by these antibodies were mapped and compared with the binding sites of murine monoclonal and human polyclonal antibodies raised against the same antigen. Methods: Two hybridomas were developed by fusing heterom yelomas to Epstein-Barr virus immortalized B cells obtained from beeke epers. The cloned hybridomas were stable and secreted up to 40 mg/L of antibody into the culture supernatant. Phospholipase A(2) specificity of the human monoclonal antibodies was confirmed by binding and inhib ition ELISA and by Western blot analysis. Epitope mapping on phospholi pase A(2) was done with the PEPSCAN method and ELISA techniques. Resul ts: The epitopes recognized by the human monoclonal antibodies were sh own to be discontinuous and did not contain the sugar residue. Similar results were obtained with polyclonal antibodies of IgG(4) isotype (f rom beekeepers) specific for phospholipase A(2), which could also inhi bit the binding of the human monoclonal antibodies to phospholipase A( 2). In contrast, antigen binding of the human monoclonal antibodies co uld not be inhibited by murine monoclonal antibodies against bee venom phospholipase A(2). Conclusions: The data indicate that the human mon oclonal antibodies obtained are representative of a part of the polycl onal immune response to phospholipase A(2) from beekeepers and may all ow a more precise analysis of the humoral immune response to phospholi pase A(2) that is associated with protection.