A Panel Set for Epitope Analysis of Myeloperoxidase (MPO)-Specific Antineutrophil Cytoplasmic Antibody MPO-ANCA Using Recombinant Hexamer Histidine-Tagged MPO Deletion Mutants |
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Authors: | Kazuo Tomizawa Eriko Mine Asami Fujii Yuko Y Ohashi Satoshi Yamagoe Yuki Hashimoto Akiko Ishida-Okawara Mie Ito Masaru Tanokura Takeki Yamamoto Yoshihiro Arimura Toshihiko Nagasawa Satoshi Mizuno Kazuo Suzuki |
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Institution: | (1) Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan;(2) The First Department of Internal Medicine, Kyorin University, School of Medicine, Tokyo, Japan;(3) Biotechnology Research Center, University of Tokyo, Tokyo, Japan;(4) Natural Products Research Department, Teikoku Hormone Mfg. Co., Ltd., Kawasaki, Japan |
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Abstract: | A major target protein of antineutrophil cytoplasmic antibody with a perinuclear staining pattern (P-ANCA) has been identified as myeloperoxidase (MPO). Recombinant deletion mutants of MPO, eight fragments of the heavy-chain subunit, and two fragments of the light chain subunit were expressed in E. coli using a pQE expression vector. The recombinant hexamer histidine-tagged fragments were partially purified as the denatured proteins on a Ni2+-charged nitrirotriacetic acid column. The recombinant fragments were reacted with a rabbit polyclonal antibody to human MPO in Western blotting. In addition, the reactivities of the proteins with MPO-ANCA-positive sera of four patients with renal diseases were examined by Western blotting. The profile of the reactivity showed that different sera recognized different sets of fragments of the heavy chain, whereas no serum reacted with the fragments of the light chain. These results indicate that the sera of patients with MPO-ANCA-positive diseases showed varied reactivities with the different fragments. Furthermore, an ELISA system using a set of the fragments completely purified by Sephacryl S-200HR column chromatography was established. The panel set is useful for subclassification of MPO-ANCA-related diseases. |
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Keywords: | Myeloperoxidase (MPO) MPO-antineutrophil cytoplasmic antibody (ANCA) epitope mapping neutrophils vascular lesion |
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