Figure 2. Characterization and Epitope Mapping of Circulating Goodpasture Autoantibodies Specific to the α3 and α5 Noncollagenous-1 Domains.

Autoantibodies were preincubated with various concentrations of the monomer α3 or α5 noncollagenous-1 (NC1) domain, and binding to immobilized antigens α3NC1 and α5NC1 was measured with the use of an enzyme-linked immunosorbent assay (ELISA). Panels A and B show means (±SE) for relative binding, expressed as a percentage of binding in the absence of NC1 monomers in solution, for α3NC1 and α5NC1 IgG antibodies, respectively, from seven patients with Goodpasture’s disease. Binding of the α3NC1 IgG antibodies to immobilized α3NC1 was strongly inhibited in the presence of soluble α3NC1 (solid circles) but not α5NC1 (open circles) (Panel A). The α5NC1 IgG antibodies had a lower affinity for α5NC1 (Panel B). Panel C shows the extent of binding of α3NC1 and α5NC1 IgG antibodies to NC1 hexamers from native glomerular basement membrane (N-GBM) and dissociated GBM (D-GBM). IgG antibodies from individual serum samples from patients with Goodpasture’s disease are represented by circles and medians by horizontal lines. Panel D shows the alignment of the α1NC1 and α5NC1 amino acid sequences corresponding to the E_A and E_B regions of the α3NC1 domain. Residues that differ from those in α1NC1 (bold) and residues that were mutated in α5 chimeras (bold red) are shown. Panel E shows means (±SE) for the inhibition of the binding of circulating α5NC1-IgG antibodies from the seven patients with Goodpasture’s disease to the α5NC1 domain. E_A-α5 chimeras are represented by solid triangles, and E_B-α5 chimeras by open triangles. The monomers α5NC1 (open circles) and α1NC1 (solid circles) were included as positive and negative controls, respectively. In Panels A, B, and E, I bars denote standard errors for seven α5NC1 antibodies.