title: Novel approach to identifying autoantibodies in rheumatoid synovitis with a biotinylated human autoantigen library and the enzyme-labeled antigen method.
abstract: Synovial tissue in rheumatoid arthritis (RA) shows dense infiltration of plasmacytes. The purpose of the present study is to identify and localize autoantibodies produced in these immunocytes in RA synovitis. We developed a novel screening system for detecting specific autoantigens. Protein antigens recognized by antibodies in the serum and synovial tissue extract from five RA patients were screened with the AlphaScreen method. For screening, a biotinylated human autoantigen library was constructed by the wheat germ cell-free protein synthesis system. The AlphaScreen analysis of 2183 proteins detected a limited number of antigens reactive with the serum and synovial tissue extract. Eighteen biotinylated proteins, containing top five showing high signals in each synovitis tissue extract, were utilized as probes for the enzyme-labeled antigen method, in order to visualize the site of specific antibody production in synovial lesions. Specific antibodies against two proteins, tripartite motif-containing 21 (TRIM21, also known as SSA/Ro52) and F-box only protein 2 (FBXO2), were visualized in the cytoplasm of plasmacytes in two RA synovitis lesions, respectively. Absorption experiments using unlabeled proteins confirmed the specificity of staining. No positive signals against these two proteins were identified in the additionally evaluated RA and osteoarthritis synovial lesions. The present study indicated 1) the usefulness of screening the human autoantigen library with the AlphaScreen assay for detecting autoantibodies in RA synovitis, and 2) the applicability of biotinylated proteins to the enzyme-labeled antigen method for visualizing the site of autoantibody production within the lesion.
authors.affiliation: Department of Pathology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Mizutani
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Matsuoka
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takeda
authors.forename: Kazuya
authors.initials: K
authors.lastname: Shiogama
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Inada
authors.forename: Kazue
authors.initials: K
authors.lastname: Hayakawa
authors.forename: Harumoto
authors.initials: H
authors.lastname: Yamada
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Miyazaki
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Tsutsumi
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D001172:Arthritis, Rheumatoid / Q000097:blood / Q000235:genetics / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001324:Autoantigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003593:Cytoplasm / Q000276:immunology / Q000378:metabolism; D044783:F-Box Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005260:Female; D015723:Gene Library; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010950:Plasma Cells / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D015203:Reproducibility of Results; D012261:Ribonucleoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013583:Synovial Membrane / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D013585:Synovitis / Q000097:blood / Q000235:genetics / Q000276:immunology*; D000099207:SS-A Antigen
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D018797:Cell Cycle Proteins; D044783:F-Box Proteins; D009419:Nerve Tissue Proteins; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C485508:FBXO2 protein, human