Characterization of autoantibodies from patients with Goodpasture's disease using a resonant mirror biosensor.
Goodpasture's disease is characterized by the binding of IgG autoantibodies to the glomerular basement membrane, leading to glomerular inflammation. The autoantigen has been identified as the noncollagenous domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). We have used the IAsys resonant mirror biosensor to analyse the extent and affinity of binding of anti-GBM antibodies from sera of patients to purified alpha3(IV) NC1. alpha3(IV) NC1 monomers were immobilized to a carboxylate cuvette, with the simultaneous use of a control well. The binding of serum from patients with Goodpasture's disease (n = 12), normal controls (n = 14) and disease controls with vasculitis (n = 14) was analysed. Antibody binding was detected in sera from all patients with Goodpasture's disease but not from controls. IAsys measurements of binding correlated with antibody levels assessed by the standardized ELISA used for clinical assays. Both ELISA and biosensor measurements showed declining antibody levels in serial serum samples from treated patients; however, the biosensor detected antibody recrudescence when ELISA remained negative. Autoantibodies from patients' serum had average affinity constants (Kd) of 6.5 x 10-11M to 52.07 x 10-10M, as determined by an inhibition assay, indicating high affinity. Sips analysis showed that the antibody response was relatively homogeneous (values of 0.46-1). Biosensor techniques can therefore be used to detect and characterize anti-GBM antibodies in serum from patients, with high sensitivity and without need for antibody purification. This technique may be useful in diagnosis and monitoring of patients with Goodpasture's disease, and may be applicable to other autoantibody mediated diseases.
Dougan
T
T
Renal Section and Department of Immunology, Faculty of Medicine, Imperial College, Hammersmith Hospital, London, UK.
Levy
J B
JB
Salama
A
A
George
A J T
AJ
Pusey
C D
CD
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D019867:Anti-Glomerular Basement Membrane Disease / Q000097:blood / Q000276:immunology*; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D015374:Biosensing Techniques / Q000379:methods; D024141:Collagen Type IV / Q000276:immunology*; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D007678:Kidney Glomerulus / Q000276:immunology; D008024:Ligands; D008297:Male; D008875:Middle Aged; D012756:Sheep
D001323:Autoantibodies; D001324:Autoantigens; D024141:Collagen Type IV; D007074:Immunoglobulin G; D008024:Ligands