Epitope analysis of the Goodpasture antigen using a resonant mirror biosensor

Abstract

SUMMARYWe have used a new technique for studying molecular interactions-a resonant mirror biosensor-to identify B cell epitopes within the Goodpasture antigen, which has recently been identified as the noncollagenous domain of the 3-chain of type IV collagen ( 3(IV)NC1). Recombinant antigen (r-3) was immobilized onto the sensing surface of a sample cuvette, and the binding of patients' autoantibodies or a MoAb to the Goodpasture antigen was followed in real time. All patients' sera bound r-3 in this system, while control sera did not bind. A MoAb inhibited the binding of all patients' autoantibodies to r-3, from 27% to 90% (mean inhibition 60%), and patients' sera cross-inhibited the binding of each other to the antigen. Binding was inhibited by pre-incubation of autoantibody with both native sheep 3(IV)NC1 and purified human 3(IV)NC1 monomers. Inhibition experiments using soluble overlapping peptides from human 3(IV)NC1 identified putative B cell epitopes. These results suggest that there is a major immunodominant epitope on the Goodpasture antigen, and that there is very limited heterogeneity in the autoantibody response in Goodpasture's disease. The resonant mirror biosensor can be successfully used to monitor antibody-antigen binding using polyclonal sera, and to map epitopes on autoantigens.

Anti-neutrophil cytoplasmic antibodies (ANCA) from patients with systemic vasculitis recognize restricted epitopes of proteinase 3 involving the catalytic site

“…IAsys is a relatively quick method of analysing binding of C‐ANCA to PR3, and so far appears highly specific. We have also used IAsys to detect binding of P‐ANCA to MPO (Griffith et al ., unpublished observations), and of anti‐glomerular basement membrane (GBM) antibodies to the Goodpasture antigen ( α 3(IV)NC1) [27]. IAsys may provide an efficient alternative to the current techniques of ELISA and indirect immunofluorescence for the detection of ANCA, particularly in situations where a result is required urgently.…”

“…Optical biosensor technology has been used to study protein interactions in real time [20–22], in particular the binding of MoAbs to their antigens [23–26]. More recently, the technique has been extended to study human antibody binding to autoantigens [27] or to immunogens [28]. In the present study we used the IAsys resonant mirror biosensor to study the binding to native PR3 of C‐ANCA from patients with active vasculitis, and of anti‐PR3 MoAbs.…”

A Self T Cell Epitope Induces Autoantibody Response: Mechanism for Production of Antibodies to Diverse Glomerular Basement Membrane Antigens

“…However, the Abs reacted only with unmasked epitopes of collagens by high concentrations of urea at very low pH, which presumably denatured the proteins (24). Several unsuccessful attempts were made to map linear B cell epitopes by synthetic peptides covering Goodpasture Ag or other collagen chains (26,27). The single exception was a mAb to Col4␣4NC1, which bound to native GBM as well as a peptide (9).…”

Circulating anti‐glomerular basement membrane autoantibodies against α3(IV)NC1 undetectable by commercially available enzyme‐linked immunosorbent assays

“…Anti‐GBM disease in the absence of circulating antibodies detected by ELISA has been reported previously. Several explanations were proposed, such as the autoantibodies targeting distinct GBM antigens other than α3(IV)NC1, 10 lower sensitivity of ELISA, 11,12 higher affinity of the antibodies towards GBM which lead them deposit in the kidney rather than present in the circulation, 13 or detection for the antibodies in the late course of disease when circulating antibodies have disappeared 14 . However, these scenarios are inadequate to explain the whole feature of the four patients in the current study.…”