Novel approach to identifying autoantibodies in rheumatoid synovitis with a biotinylated human autoantigen library and the enzyme-labeled antigen method

Yasuyoshi Mizutani, Kazuhiro Matsuoka, Hiroyuki Takeda, Kazuya Shiogama, Ken ichi Inada, Kazue Hayakawa, Harumoto Yamada, Tatsuhiko Miyazaki, Tatsuya Sawasaki, Yaeta Endo, Yutaka Tsutsumi

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)57-70
Number of pages14
JournalJournal of Immunological Methods
Volume387
Issue number1-2
DOIs
Publication statusPublished - 31-01-2013

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Synovitis
Autoantigens
Autoantibodies
Rheumatoid Arthritis
Antigens
Enzymes
Tissue Extracts
Proteins
Plasma Cells
F-Box Proteins
Antibodies
Serum
Germ Cells
Osteoarthritis
Triticum
Antibody Formation
Cytoplasm
Staining and Labeling

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology

Cite this

Mizutani, Yasuyoshi ; Matsuoka, Kazuhiro ; Takeda, Hiroyuki ; Shiogama, Kazuya ; Inada, Ken ichi ; Hayakawa, Kazue ; Yamada, Harumoto ; Miyazaki, Tatsuhiko ; Sawasaki, Tatsuya ; Endo, Yaeta ; Tsutsumi, Yutaka. / Novel approach to identifying autoantibodies in rheumatoid synovitis with a biotinylated human autoantigen library and the enzyme-labeled antigen method. In: Journal of Immunological Methods. 2013 ; Vol. 387, No. 1-2. pp. 57-70.
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Novel approach to identifying autoantibodies in rheumatoid synovitis with a biotinylated human autoantigen library and the enzyme-labeled antigen method. / Mizutani, Yasuyoshi; Matsuoka, Kazuhiro; Takeda, Hiroyuki; Shiogama, Kazuya; Inada, Ken ichi; Hayakawa, Kazue; Yamada, Harumoto; Miyazaki, Tatsuhiko; Sawasaki, Tatsuya; Endo, Yaeta; Tsutsumi, Yutaka.

In: Journal of Immunological Methods, Vol. 387, No. 1-2, 31.01.2013, p. 57-70.

Research output: Contribution to journalArticle

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