Mapping B cell epitopes in Goodpasture's disease.
The target antigen of the pathogenic autoantibodies in Goodpasture's disease is the noncollagenous domain of the alpha 3 chain of type IV collagen. A panel of membrane-bound peptides was used to identify antibody-binding regions within the protein, and the significance of the binding by inhibition studies using soluble peptides, and by a structural analysis of the antigen, was confirmed. A total of 117 overlapping 12-mer peptides spanning the entire antigen was simultaneously synthesized as individual spots on a cellulose membrane. All nine patients' sera bound to the membrane, with a conserved pattern of peptides recognized by all sera. Inhibition studies were performed using a panel of overlapping 20-mer peptides, also spanning the entire antigen. Peptides from the regions that bound IgG were able to inhibit the binding of autoantibodies to native antigen. Predictions of the secondary structure of the noncollagenous domain of the alpha 3 chain of type IV collagen were performed by a conventional hydropathy plot and by multiple alignment of homologous alpha chains of type IV collagen and comparison with a structural data base. The core peptides binding and inhibiting Goodpasture's antibodies were predicted to be surface exposed and antigenic. Thus, the conformational epitope(s) of the Goodpasture antigen can be mapped using linear peptides.
Levy
J B
JB
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom.
Coulthart
A
A
Pusey
C D
CD
D000595:Amino Acid Sequence; D019867:Anti-Glomerular Basement Membrane Disease / Q000097:blood / Q000276:immunology*; D001323:Autoantibodies / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000276:immunology*; D002478:Cells, Cultured; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006801:Humans; D007074:Immunoglobulin G / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology / Q000494:pharmacology; D012680:Sensitivity and Specificity
D001323:Autoantibodies; D007074:Immunoglobulin G; D010455:Peptides