The goal of the present proposal is to establish the amino acid sequences of the antigenic determinants of the Ku-antigen involved in autoimmune diseases. The Ku-antigen has been characterized as a DNA binding protein complex of two polypeptides, 86/70 kD. It is recognized by sera from patients with scleroderma and systemic lupus erythematosus. The prerequisites for the proposed experiment are: 1) monoclonal antibodies to the antigen have been developed; 2) the protein complex has been purified and characterized, and 3) cDNA clones expressing epitopes of the antigen have been cloned and partially sequenced. The number of the epitopes recognized by the monoclonal antibodies will be determined and mapped on the protein complex. This will provide the basis for analysis of the binding specificities of human autoantibodies. Sera from patients with autoimmune diseases will be screened for antibodies to the antigen using purified antigen in ELISA, immunoblot and radioimmunoassays. The epitopes recognized by the autoimmune sera will be determined in competition experiments with the monoclonal antibodies. These epitopes will be mapped on the antigen by using recombinant DNA technology and the monoclonal antibodies. The amino acid sequences of the epitopes will be derived from the cDNA clones and sublibraries of their fragments constructed in expression vectors. Alternatively, antigenic peptides produced by proteolytic degradation of the antigen will be purified and sequenced. These experiments will allow us to investigate the possible correlations between the epitope spectrum and the clinical parameters during the development of the autoimmune diseases. The established amino acid sequences can be used for developing synthetic peptides as potential diagnostic probes.
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