? Human idiopathic membranous glomerulonephritis (MGN) is assumed to be an organ-specific autoimmune disease of the kidney in which disease-inducing autoantibodies are directed against an autoantigen located on the surface of podocytes. However, to date no autoantibodies have been detected in patients' sera and the identity of the autoantigen remains unknown. Thus this important area of research remains at a standstill. The possibility that autoantibodies are quickly absorbed by the kidney and subsequently abate to low or undetectable levels when patients first present with full-blown nephrotic syndrome is supported by similar observation in Heymann nephritis, a rat model of MGN. We propose to obtain evidence for the existence of these autoantibodies by a novel approach: screening of phage display antibody libraries of patients with idiopathic MGN. Phage display provides several advantages over sera; (1) expression of the antibody repertoire is independent of any regulatory mechanisms that may affect expression of serum antibodies, (2) phage particles binding to rare antigens in tissue sections can be distinguished from residual normal IgG, (3) phage antibodies that bind to the selective substrate can be recovered and amplified and (4) monoclonal antibody fragments can be readily obtained and sequenced. Phage-displayed antibodies will be used for staining of human kidney sections. Libraries will be enriched for specific phage antibodies and monoclonal antibody fragments will be selected and expressed. The selected clones will be used to isolate and identify the autoantigen from renal homogenates by immunoprecipitation, western blot analysis and N-terminal microsequencing. Peptide ligands for the high affinity clones will be selected from random peptide phage libraries and sequenced to define a consensus sequence, which could identify potential autoantigens in the protein database. Identification of the autoantigen of human MGN will lead to renewed efforts to delineate the mechanism of pathogenesis and provide reagents and data to support further clinical and preclinical studies. Human monoclonal antibodies against the antigen or its fragments will be useful for in vitro studies or development of a passive model that more closely mimics the human disease. Knowledge of the renal antigen will assist in the development of a clinical diagnostic test and potentially in new approaches to therapy or prevention of MGN ? ?
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