Antibody competition studies with gold-labelling immunoelectron microscopy.

Competitive studies, involving neutralizing monoclonal antibodies (NTmAbs) to outercoat proteins of Akabane virus were performed by immunoelectron microscopy. The experiments were designed to determine whether the NTmAbs were directed against the same or spatially different epitopes. Characteristics of NTmAbs in direct and indirect gold-labelling studies were determined. It was found that the protein A method gave cross-contamination of the immuno-gold complexes whereas direct conjugation of the NTmAbs to gold probes gave clean, specific and intense labelling. Analysis of dilution curves confirmed that saturation of antigenic sites did not occur and secondly determined the optimum working dilutions for the conjugated probes. The data generated in the preliminary studies enabled reliable results to be obtained from the double-labelling competitive experiment. We found that the 2 NTmAbs were directed to either the same epitope or to 2 separate but neighbouring epitopes where the binding of one NTmAb inhibited the binding of the second. The results demonstrate that if reliable data is to be obtained in double-labelling immunoelectron microscopical studies then experiments must be meticulously designed.