title: Circulating anti-glomerular basement membrane autoantibodies against α3(IV)NC1 undetectable by commercially available enzyme-linked immunosorbent assays.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Renal Division, Department of Medicine, Peking University First Hospital, Institute of Nephrology, Peking University, Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China.
authors.forename: Xiao-Yu
authors.initials: XY
authors.lastname: Jia
authors.forename: Zhen
authors.initials: Z
authors.lastname: Qu
authors.forename: Zhao
authors.initials: Z
authors.lastname: Cui
authors.forename: Rui
authors.initials: R
authors.lastname: Yang
authors.forename: Juan
authors.initials: J
authors.lastname: Zhao
authors.forename: Ming-Hui
authors.initials: MH
authors.lastname: Zhao
mesh_terms: D000328:Adult; D019867:Anti-Glomerular Basement Membrane Disease / Q000097:blood / Q000276:immunology* / Q000473:pathology; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology*; D001706:Biopsy; D015153:Blotting, Western; D002681:China; D024141:Collagen Type IV / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007668:Kidney / Q000276:immunology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D011237:Predictive Value of Tests; D011487:Protein Conformation; D012680:Sensitivity and Specificity
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D024141:Collagen Type IV; D000939:Epitopes; C051951:antiglomerular basement membrane antibody; C057112:type IV collagen alpha3 chain