A PANEL SET FOR EPITOPE ANALYSIS OF MYELOPEROXIDASE (MPO)-SPECIFIC ANTINEUTROPHIL CYTOPLASMIC ANTIBODY MPO-ANCA USING RECOMBINANT HEXAMER HISTIDINE-TAGGED MPO DELETION MUTANTS

A major target protein of antineutrophil cytoplasmic antibody with a p erinuclear staining pattern (P-ANCA) has been identified as myeloperox idase (MPO). Recombinant deletion mutants of MPO, eight fragments of t he heavy-chain subunit, and two fragments of the light chain subunit w ere expressed in E. coli using a pQE expression vector. The recombinan t hexamer histidine-tagged fragments were partially purified as the de natured proteins on a Ni2+-charged nitrirotriacetic acid column. The r ecombinant fragments were reacted with a rabbit polyclonal antibody to human MPO in Western blotting. In addition, the reactivities of the p roteins with MPO-ANCA-positive sera of four patients with renal diseas es were examined by Western blotting. The profile of the reactivity sh owed 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 fragm ents. Furthermore, an ELISA system using a set of the fragments comple tely purified by Sephacryl S-200HR column chromatography was establish ed. The panel set is useful for subclassification of MPO-ANCA-related diseases.