Epitope analysis of the Goodpasture antigen using a resonant mirror biosensor.
We have used a new technique for studying molecular interactions-a resonant mirror biosensor-to identify B cell epitopes within the Goodpasture antigen, which has recently been identified as the non-collagenous domain of the alpha 3-chain of type IV collagen (alpha 3(IV)NC1). Recombinant 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 patients' sera bound r-alpha 3 in this system, while control sera did not bind. A MoAb inhibited the binding of all patients' autoantibodies to r-alpha 3, from 27% to 90% (mean inhibition 60%), and patients' sera cross-inhibited the binding of each other to the antigen. Binding was inhibited by pre-incubation of autoantibody with both native sheep alpha 3(IV)NC1 and purified human alpha 3(IV)NC1 monomers. Inhibition experiments using soluble overlapping peptides from human alpha 3(IV)NC1 identified putative B cell epitopes. These results suggest that there is a major immunodominant epitope on the Goodpasture antigen, and that there is very limited heterogeneity in the autoantibody response in Goodpasture's disease. The resonant mirror biosensor can be successfully used to monitor antibody-antigen binding using polyclonal sera, and to map epitopes on autoantigens.
Levy
J B
JB
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
Turner
A N
AN
George
A J
AJ
Pusey
C D
CD
D000595:Amino Acid Sequence; D019867:Anti-Glomerular Basement Membrane Disease / Q000276:immunology*; D001324:Autoantigens / Q000032:analysis*; D015374:Biosensing Techniques; D003094:Collagen / Q000032:analysis*; D024141:Collagen Type IV; D018604:Epitope Mapping; D006801:Humans; D016056:Immunodominant Epitopes / Q000032:analysis*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis
D001324:Autoantigens; D024141:Collagen Type IV; D016056:Immunodominant Epitopes; D011994:Recombinant Proteins; C057112:type IV collagen alpha3 chain; D003094:Collagen