Development and application of an ELISA for Goodpasture's disease based on sheep alpha 3(IV)NC1 domains.
A high titre of antibodies to collagenase-solubilised human glomerular basement membrane (CS-GBM) is almost pathognomic of Goodpasture's (anti-GBM) disease. In order to develop an assay independent of scarce human material, a molecule of approximately 26 kD corresponding to the C-terminal NC1 domain of the alpha 3 chain of type IV collagen was purified from sheep GBM by gel filtration and reverse phase HPLC. This molecule was antigenic when assessed by inhibition studies, by immunoblotting, and as a ligand on ELISA plates. An ELISA using this sheep alpha 3(IV)NC1 preparation to detect circulating anti-GBM antibodies gave comparable results to the standard RIA using crude CS-GBM. Sera from patients with a variety of nephropathies other than Goodpasture's disease gave negative results. In a prospective study, 170 consecutive sera were analysed by both the ELISA and the RIA. Twenty seven specimens gave positive results in one or both of the assays. Eleven of these were confirmed as true positive results and all were correctly identified by the RIA. Two false negative results in the ELISA occurred in previously treated cases, and both sera were only weakly positive by RIA. The RIA gave 13 false positive results compared with five by ELISA. The ELISA using highly purified sheep antigen is a robust, reliable, and more specific alternative to immunoassays based on crude human antigen preparations.
Katbamna
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I
Dept. of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
Turner
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N
Ross
C N
CN
Pusey
C D
CD
D000818:Animals; D019867:Anti-Glomerular Basement Membrane Disease / Q000175:diagnosis* / Q000276:immunology; D000941:Antigens / Q000737:chemistry* / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology; D001485:Basement Membrane / Q000276:immunology; D003094:Collagen / Q000737:chemistry* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007678:Kidney Glomerulus / Q000276:immunology; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012756:Sheep
D000941:Antigens; D001323:Autoantibodies; D008024:Ligands; D010446:Peptide Fragments; D003094:Collagen