A library of monoclonal antibodies to Escherichia coli K-12 pyruvate dehydrogenase complex. Competitive epitope...

Presented here are competitive epitope mapping studies on a monoclonal antibody library to K-12 Escherichia coli pyruvate dehydrogenase complex (PDHc) and its pyruvate decarboxylating (EC1.2.4.1) subunit (E1). Several of the monoclonal antibodies had been found to inhibit PDHc from 0 to 98%. Of the 10 monoclonal antibodies that showed the greatest inhibition of PDHc, 4 were elicited by PDHc and 6 by E1. Surface plasmon resonance was used for competitive epitope mapping and revealed that these 10 monoclonal antibodies had at least 6 separate binding regions on the PDHc. The three monoclonal antibodies that demonstrated the strongest inhibition appeared to bind the same region on the PDHc. Mapping studies with the E1 antigen using an additional five monoclonal antibodies demonstrated that the two strongest inhibitory monoclonal antibodies (18A9 and 21C3) shared the same binding region on E1, whereas the third strongest inhibitor (15A9) displayed an epitope region that overlapped the previous two on the E1 subunit. Antibody 15A9 had been shown to counteract GTP regulation of PDHc. Simultaneous multiple site binding experiments confirmed that the defined epitope regions were indeed independent. Limited competitive epitope binding experiments using radiolabeled E1 confirmed the surface plasmon resonance results.