EPITOPE ANALYSIS OF THE GOODPASTURE ANTIGEN USING A RESONANT MIRROR BIOSENSOR

We have used a new technique for studying molecular interactions-a res onant mirror biosensor-to identify B cell epitopes within the Goodpast ure antigen, which has recently been identified as the non-collagenous domain of the alpha 3-chain of type IV collagen (alpha 3(IV)NC1). Rec ombinant antigen (r-alpha 3) was immobilized onto the sensing surface of a sample cuvette, and the binding of patients' autoantibodies or a MoAb to the Goodpasture antigen was followed in real time. All patient s' sera bound r-alpha 3 in this system, while control sera did not bin d. A MoAb inhibited the binding of all patients' autoantibodies to r-a lpha 3, from 27% to 90% (mean inhibition 60%), and patients' sera cros s-inhibited the binding of each other to the antigen. Binding was inhi bited by pre-incubation of autoantibody with both native sheep alpha 3 (IV)NC1 and purified human alpha 3(IV)NC1 monomers. Inhibition experim ents using soluble overlapping peptides from human alpha 3(IV)NC1 iden tified putative B cell epitopes. These results suggest that there is a major immunodominant epitope on the Goodpasture antigen, and that the re is very limited heterogeneity in the autoantibody response in Goodp asture's disease. The resonant mirror biosensor can be successfully us ed to monitor antibody-antigen binding using polyclonal sera and to ma p epitopes on autoantigens.