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Protein microarray characterization of autoantibody responses in rheumatoid arthritis and systemic lupus erythematosus

In rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and other autoimmune rheumatic diseases, our understanding of the specificity of the autoantibody response and the role of autoantibodies in pathogenesis are limited. We developed antigen microarray technology to perform multiplex characterization of autoantibody responses. Antigen microarrays are produced by attaching thousands of proteins and peptides to addressable locations on the surface of solid supports using a robotic arrayer. Arrays are probed with serum from disease and control patients, followed by anti-human secondary antibodies covalently-conjugated to spectrally-resolvable fluorochromes. We initially developed a 'connective tissue disease' array containing structurally diverse autoantigens including nucleic acids, histones, hnRNPs, snRNPs, collagens, Ro, La, SCL-70, CENP-B, Jo-1, pyruvate dehydrogenase, and post-translationally-modified antigens. Array analysis of serum derived from patients with SLE, rheumatoid arthritis, Sjögren's syndrome, mixed connective tissue diseases, scleroderma, myositidies, and primary biliary cirrhosis identified autoantibody response patterns characteristic of these diseases (Fig. 1). We are now developing 'synovial proteome' arrays to study the autoantibody responses in RA, and have produced first-generation arrays containing 450 distinct protein and peptide candidate antigens derived from synovial joints. These include native and citrulline-modified filaggrin peptides, native and deiminated fibrinogen and vimentin, glucose-6-phosphate isomerase, collagen types I, II, III, IV and V, hnRNP-A2/RA33, hnRNP-D, immunoglobulins, HCgp39, BiP, and HSPs 60, 65, 70, and 90. We probe our arrays with sera and synovial fluid from RA and control patients. We are using our arrays to examine serial serum samples from patients with early RA to: (1) identify antigens targeted early in disease, and (2) examine for evidence of B-cell epitope spreading. 'Synovial proteome' arrays represent a powerful tool to study the breadth and specificity of autoreactive B-cell responses, and to identify candidate and define relevant autoantigens in RA.

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Robinson, W., Huber, W., van Venrooij, W. et al. Protein microarray characterization of autoantibody responses in rheumatoid arthritis and systemic lupus erythematosus. Arthritis Res Ther 4 (Suppl 1), 76 (2002).

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