High affinity of anti-GBM antibodies from Goodpasture and transplanted Alport patients to α3(IV)NC1 collagen

Background. Anti-glomerular basement membrane (anti-GBM) antibody-mediated diseases are characterized by rapidly progressive glomerulonephritis (RPGN) that often results in irreversible loss of renal function and renal failure. Although many factors contribute to the fulminant nature and treatment resistance of this disease, we questioned whether high affinity autoantibody-alpha 3(IV) collagen interactions lead to persistent antibody deposition, thereby perpetuating inflammation. To address this hypothesis, the binding kinetics of human anti-GBM antibodies (Ab) to alpha 3(IV)NCl were evaluated using an optical biosensor interaction analysis. Methods. Polyclonal anti-CBM Abs were purified by alpha 3(IV)NCl affinity chromatography from the sera of patients with anti-GEM AB-mediated diseases, including individuals with Goodpasture syndrome (GS), idiopathic RPGN (N = 7), and Alport syndrome (AL) following kidney transplantation (N = 4). The affinity-binding characteristics of the autoantibodies were determined using a biosensor analysis system, with immobilized bovine alpha 3(IV)NCl dimers. Results. All of the autoantibody preparations bound to alpha 3(IV)NCl, whereas none bound to alpha 1(IV)NCl (control). Purified, normal serum IgG did not bind to either antigen. Estimated dissociation constants (Kd) for the purified autoantibodies were 1.39E-04 +/- 7.30E-05 s(-1) (GS) and 8.90E-05 +/- 2.80E-05 s(-1) (AL). Their estimated association constants (K-a) were 2.67E + 04 +/- 1.8E + 04 (M-Is-1) and 2.76E +/- 04 +/- 1.70E + 04 (M-Is-1) for GS and AL patients, respectively. By comparison with other nb interactions, these Abs demonstrated high affinity, with relatively high on (binding) rates and slow off (dissociation) rates. Conclusions. The results suggest that anti-GEM Abs bind rapidly and remain tightly bound to the GBM in vivo. This property likely contributes to both the fulminant nature of this disease and its resistance to therapy, because persistent glomerular Ab deposition has the potential to produce continuous inflammation, despite removal of circulating Abs and adequate immuno-suppression.