Circulating anti‐glomerular basement membrane autoantibodies against α3(IV)NC1 undetectable by commercially available enzyme‐linked immunosorbent assays

Abstract

Cases with anti-glomerular basement membrane (GBM) disease have been reported with linear deposit of immunoglobulin G (IgG) along GBM, but have undetectable anti-GBM antibodies in circulation by enzyme linked immunosorbent assays (ELISA). We speculated that the structure of the antigens recognized by these antibodies may contribute to the negative results of ELISA. Sera from four patients were collected, with typical linear deposit of IgG along GBM but no anti-GBM reactivity by commercial ELISA kits. Circulating anti-GBM antibodies were detected by indirect immunofluorescence. Antigen specificity and its conformational structure was investigated by western-blot analysis, using recombinant human α1-α5(IV)NC1 and chimeric proteins E(A) and E(B) as antigens. The presence of circulating anti-GBM antibodies were confirmed by indirect immunofluorescence with linear deposit of IgG towards cryptic epitopes along GBM on normal kidney sections. These antibodies did not recognize recombinant human α1, α2, α4 or α5(IV)NC1, but could blot α3(IV)NC1 under non-reducing non-boiling conditions on western-blot analysis, when the conformational epitope(s) on α3(IV)NC1 were thought to be preserved. When α3(IV)NC1 was prepared under reducing conditions with β-mercaptoethanol and/or boiled to destroy the disulfide bonds, the binding with the antibodies disappeared. Moreover, these antibodies recognized neither E(A) nor E(B) , indicating their distinct epitope repertoire. Circulating anti-GBM antibodies undetectable by ELISA could recognize cryptic and conformation-dependent epitopes restricted on α3(IV)NC1, distinct from E(A) and E(B) . Indirect immunofluorescence was necessary for antibody detection and treatment monitoring under such circumstances.