A LIBRARY OF MONOCLONAL-ANTIBODIES TO ESCHERICHIA-COLI K-12 PYRUVATE-DEHYDROGENASE COMPLEX - COMPETITIVE EPITOPE MAPPING STUDIES

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