A SINGLE-CHAIN FV REACTIVE WITH THE GOODPASTURE ANTIGEN

Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapid ly progressive glomerulonephritis and pulmonary hemorrhage. P1, a muri ne monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NCI), has- been a valuable reagent in investigating the pathogenesis of this diso rder. The purpose of this study was to generate and characterize a rec ombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA enco ding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The p urified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expres sed product. HBR-3 was shown by ELISA, immunoblotting, and immunohisto logic techniques, to have the same specificity for alpha 3(IV)NC1 as P 1 and autoantibodies from patients with Goodpasture's disease. HBR-3 a nd P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular bas ement membrane and distal tubular basement membrane. It did not bind t o the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigeni c form. We have, therefore, generated a scFv which reproduces the spec ific binding properties of the parent monoclonal antibody, P1. The pot ential of HBR-9 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should perm it new approaches to the investigation of Goodpasture's disease.