Goodpasture syndrome is an autoimmune disorder in humans characterized by rapidly progressive glomerulonephritis that is often accompanied by pulmonary hemorrhage. This fatal disease is mediated by antibodies directed against the carboxyl-terminal non-collagenous domain (NC1) of a novel collagen IV chain, designated a3(IV). Several lines of evidence indicate that the epitope(s) involved in the autoimmune response is(are) specific and restricted to a unique, strongly conserved and highly immunogenic motif within the a3(IV) NC1 domain. The objectives of this proposal are to characterize the Goodpasture antigen [as(IV) NC1 domain] using recombinant DNA techniques and to produce recombinant antigen for diagnostic and therapeutic testing. Clones will be initially identified in a specific expression library using antibodies directed against the NC1 domain of a3(IV). Those covering the full length of the Goodpasture antigen will be used to generate immortalized cell lines expressing recombinant antigen. Specific proteolysis of recombinant antigen combined with Western blot studies using Goodpasture serum will be performed to define proteolytic fragments that maintain reactivity. Full- size Goodpasture antigen or antigenic fragments thereof will be used in an attempt to reproduce the disorder in animals and to localize and delimit the antigenic motif. The recombinant antigen will be employed to develop an easy, reliable and inexpensive diagnostic ELISA test while, based on the success of obtaining an animal model, specific reactive fragments will be tested for their effectiveness in blocking circulating antibodies without inducing an immunological response (blocking haptens).
