Development of high-sensitive periodontitis diagnosis method using biotinylated polysaccharide antigens

利用生物素化多糖抗原开发高灵敏牙周炎诊断方法

• 批准号: 05454563
• 负责人: KOGA Toshihiko
• 金额: $ 4.22万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454563/
• 关键词: Periodontal disease; Polysaccharide antigen; Diagnosis method; Periodontopathic bacteria; Biotin; ELISA; 単球; サイトカイン

Periodontitis has been recently considered to be an infectious disease that is induced by several specific bacteria. In this study, we developed a rapid and specific method for measuring antibodies to periodontopathic bacteria. Serotype-specific polysaccharide antigens (SPAs) were extracted from whole cells of Actinobacillus actinomycetemcomitans by autoclaving and purified by chromatography. The purified SPAs were activated with cyanogen bromide and then treated with adipic acid dihydrazide. The preparations were coupled with biotin or bovine serum albumin (BSA) by the carbodiimide method. Enzyme-linked immunosorbent assay plates pretreated with avidin were allowed to react with biotinylated SPAs. Studies using rabbit anti-A.a.sera showed the assay utilizing avidin-biotin was very specific and sensitive. Antibodies to the antigens in 1 : 6,44 to 1 : 12,800 dilutions of human sera from subjects with periodontitis could be detected by using the assay. Subcutaneous immunization of rabbits with BSA-SPA induced high titers of serum anti-SPA antibodies, and these antibodies enhanced phagocytosis and killing of A.a.by human polymorphonuclear leukocytes. These findings suggest that proteinconjugated polysaccharide antigens may be useful for diagnosis of periodontal disease and elucidating an etiological role of the antigens in the induction of the infectious disease.

牙周炎最近被认为是一种由几种特定细菌引起的传染病。在这项研究中，我们开发了一种快速和特异性的方法来检测牙周病细菌的抗体。采用高压灭菌法从放线菌comitans全细胞中提取血清型特异性多糖抗原，并进行层析纯化。纯化后的spa用溴化氰活化，然后用己二酸二肼处理。用碳二亚胺法与生物素或牛血清白蛋白（BSA）偶联。允许用亲和素预处理的酶联免疫吸附测定板与生物素化的spa反应。兔抗a.a的研究。血清显示，利用亲和素-生物素的检测具有很高的特异性和敏感性。该方法可检测到1∶6,44 ~ 1∶12,800稀释度的牙周炎患者血清抗原抗体。兔皮下接种BSA-SPA可诱导血清抗spa抗体的高滴度，这些抗体增强了人多形核白细胞对a.a.a的吞噬和杀伤。这些发现表明，蛋白结合多糖抗原可能有助于牙周病的诊断，并阐明抗原在感染性疾病诱导中的病因学作用。

项目成果

M.Kawasaki 他5名: "An enzyme-linked immunosorbent assay for measuring antibodies to serotype-specific polysaccharide antigens of Actinobacillus actinomycetemcomitans" Journal of Microbiological Methods. (印刷中). (1995)

M. Kawasaki 和其他 5 人：“用于测量放线杆菌伴放线杆菌血清型特异性多糖抗原的酶联免疫吸附测定法”《微生物学方法杂志》（1995 年）。

Y.Nakano et al.: "Molecular and immunological characterization of a 64-kDa protein of Actinobacillus actinomycetemcomitans." Oral Microbiol.Immunol.(in press). (1995)

Y.Nakano 等人：“Actinobacillus actinomycetemcomitans 的 64-kDa 蛋白质的分子和免疫学特征。”

M.Kawasaki et al.: "An enzyme-linked immunosorbent assay for measuring antibodies to serotype-specific polysaccharide antigens of Actinobacillus actinomycetemcomitans." J.Microbiol.Methods. (in press). (1995)

M.Kawasaki 等人：“一种酶联免疫吸附测定法，用于测量伴放线放线杆菌血清型特异性多糖抗原的抗体。”

T.Koga 他6名: "The 64-kDa GroEL-like protein of Actinobacillus actinomycetemcomitans" Journal of Periodontal Research. 28. 47-477 (1993)

T. Koga 和其他 6 人：“Actinobacillus actinomycetemcomitans 的 64-kDa GroEL 样蛋白”，《牙周研究杂志》28. 47-477 (1993)。

T.Nishihara: "Membrane-associated interleukin-1 Promotes Osteoclast-like cell formation in vitro" Bone Mineral. 25. 15-24 (1994)

T.Nishihara：“膜相关白细胞介素 1 促进体外破骨细胞样细胞形成”骨矿物质。