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

牙周炎是一种由几种特定细菌引起的感染性疾病。在这项研究中，我们开发了一种快速和特异性的方法来测量牙周病细菌的抗体。用高压灭菌法从伴放线放线杆菌全细胞中提取血清型特异性多糖抗原（SPAs），并经层析纯化。纯化的SPA用溴化氰活化，然后用己二酸二酰肼处理。制备物通过碳二亚胺法与生物素或牛血清白蛋白（BSA）偶联。使用抗生物素蛋白预处理的酶联免疫吸附测定板与生物素化SPA反应。用兔抗A.a.血清进行的研究表明，利用亲和素-生物素的检测是非常特异和敏感的。用该方法可检测1：6.44 ~ 1：12，800稀释度的牙周炎患者血清中的抗体。用BSA-SPA皮下免疫兔子诱导高滴度的血清抗SPA抗体，并且这些抗体增强吞噬作用和对人多形核白细胞的杀伤。A.a.by这些研究结果表明，蛋白结合多糖抗原可能是有用的牙周病的诊断和阐明的病原学作用的抗原诱导的感染性疾病。

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.