DEVELOPMENT OF LATEX AGGLUTINATION TEST FOR SPECIFIC DETECTION OF BACTEROIDES GINGIVALIS

本研究的目的是建立一种检测牙龈拟杆菌菌毛的胶乳凝集试验。本试验可用于牙菌斑及龈沟液中龈芽胞杆菌菌毛抗原的检测，为牙周病的诊断提供依据。从新鲜生长的牙龈芽孢杆菌381细胞中机械分离菌毛，用DEAE Sepharose Fast Flow柱层析纯化菌毛。制备了兔多克隆抗体（pAb）和鼠单克隆抗体（mab），用于乳胶颗粒的致敏。单抗的同型分别为IgM和IgGl。乳胶颗粒的敏化是通过在连续稀释的抗体溶液中孵育完成的。在玻片上进行凝集试验，并根据凝集形成的快速性和程度对结果进行分级（-、+、++、+++）。经pAb致敏的乳胶颗粒能使所有的牙龈芽孢杆菌凝集，而经mab致敏的乳胶颗粒仅能凝集11株牙龈芽孢杆菌中的8株。pAb-或mab -致敏乳胶颗粒与其他拟杆菌种类的细胞均不发生反应，包括黑素芽孢杆菌、中间芽孢杆菌、牙髓芽孢杆菌、溶糖芽孢杆菌和结核芽孢杆菌。用单抗或单抗致敏的乳胶颗粒在添加毛蛋白时可被凝集，但不包括LPS或牙龈芽孢杆菌381的荚膜物质。此外，发现pAb和mab致敏乳胶制剂在检测毛纤维抗原方面同样敏感（毛纤维的最小凝集剂量，每次测试4 ng）。我们的研究结果表明，这些抗体可能有助于制备牙周病的免疫诊断试剂盒。

The purpose of this study was to develop a latex agglutination test to detect the fimbriae of Bacteroides gingivalis cells. This test can be applied to demonstrate fimbrial antigen of B. gingivalis in dental plaque and gingival crevicular fluid for possible diagnosis of periodontal disease.The fimbriae were mechanically detached from freshly grown cells of B. gingivalis 381 and purified by a DEAE Sepharose Fast Flow column chromatography. Rabbit polyclonal antibody (pAb) and murine monoclonal antibodies (mAbs) to the fimbriae were prepared and were used for sensitization of latex particles. The isotype of mAb was found to be IgM or IgGl, respectively. Sensitization of latex particles was done by incubating them in serially diluted antibody solutions. The agglutination test was carried out on a slide glass and the results were graded (-, +, ++, +++) according to the promptness and extent of the aggregate formation. The latex particles sensitized with pAb agglutinated all B. gingivalis strains tested, while mAb-sensitized particles agglutinated only 8 out of 11 strains tested. Neither pAb- nor mAb-sensitized latex particles reacted with cells of other Bacteroides species including B. melaninogenicus, B. intermedius, B. endodontalis, B. assacharolyticus and B. buccae. Latex particles sensitized with pAb or mAb were agglutinated upon addition of the fimbrial protein, but not LPS or capsular substance of B. gingivalis 381. Furthermore, it was found that both pAb- and mAb-sensitized latex preparations were equally sensitive in detecting the fimbrial antigen (minimum agglutinating dose of fimbriae, 4 ng per test). Our findings indicate that these antibodies may be useful for the preparation of an immunodiagnostic kit for periodontal disease.