Human monoclonal or polyclonal antibodies recognize predominantly discontinuous epitopes on bee venom phospholipase...

Two hybridomas, which secrete human monoclonal antibodies of IgG4 isotype specific for the main bee venom antigen/allergen phospholipase A2, 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.

Two hybridomas were developed by fusing heteromyelomas to Epstein-Barr virus immortalized B cells obtained from beekeepers. The cloned hybridomas were stable and secreted up to 40 mg/L of antibody into the culture supernatant. Phospholipase A2 specificity of the human monoclonal antibodies was confirmed by binding and inhibition ELISA and by Western blot analysis. Epitope mapping on phospholipase A2 was done with the PEPSCAN method and ELISA techniques.

The epitopes recognized by the human monoclonal antibodies were shown to be discontinuous and did not contain the sugar residue. Similar results were obtained with polyclonal antibodies of IgG4 isotype (from beekeepers) specific for phospholipase A2, which could also inhibit the binding of the human monoclonal antibodies to phospholipase A2. In contrast, antigen binding of the human monoclonal antibodies could not be inhibited by murine monoclonal antibodies against bee venom phospholipase A2.

The data indicate that the human monoclonal antibodies obtained are representative of a part of the polyclonal immune response to phospholipase A2 from beekeepers and may allow a more precise analysis of the humoral immune response to phospholipase A2 that is associated with protection.