title: Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions.
abstract: In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders.
authors.affiliation: Department of Pathology, Fujita Health University School of Medicine , Toyoake, Aichi 470-1192, Japan.
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Mizutani
authors.affiliation: Department of Pathology, Fujita Health University School of Medicine , Toyoake, Aichi 470-1192, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Shiogama
authors.affiliation: Department of Pathology, Fujita Health University School of Medicine , Toyoake, Aichi 470-1192, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Onouchi
authors.affiliation: Department of Pathology, Banbuntane Houtokukai Hospital, Fujita Health University School of Medicine , Nakagawa-ku, Nagoya, Aichi 454-8509, Japan.
authors.forename: Kouhei
authors.initials: K
authors.lastname: Sakurai
authors.affiliation: Department of Pathology, Banbuntane Houtokukai Hospital, Fujita Health University School of Medicine , Nakagawa-ku, Nagoya, Aichi 454-8509, Japan.
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Inada
authors.affiliation: Department of Pathology, Fujita Health University School of Medicine , Toyoake, Aichi 470-1192, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Tsutsumi
keywords: AlphaScreen method; biotinylated antigen; enzyme-labeled antigen method; plasma cell; wheat germ cell-free protein synthesis system