Development of diagnostic system in periodontal disease using synthetic peptides containing epitopes of surface protein of periodontopathic bacteria

牙龈卟啉单胞菌是一种厌氧革兰氏阴性杆菌，常从牙周病患者的牙周袋中分离出来。该菌已被认为是成人牙周炎发展的主要病原体。用醋酸纤维纸尖酶联免疫吸附试验检测牙周炎假单胞菌(P.G.)并对其在龈下菌斑标本中的抗体反应进行了研究。利用多针多肽合成技术，合成了多个序列重叠的多肽。在41K菌毛和72-CSK蛋白分子中分别检测到6个和7个与牙周病患者血清有明确反应的免疫优势区。获得了抗41K菌毛的兔多克隆抗体(Poly PGF-I)和鼠单抗(mAbPGF-I)。多聚前列腺素F-I和单抗前列腺素F-I与20,000-2,000,000个P.G.细胞组成的细胞悬液通过ELISA方法反应。根据Pg细胞的数量，可将显影纸尖分为+、+、++、+四个等级。Poly PGF-I和mAbPGF-I能与P.G.细胞反应，而这两种抗体都不能与其他口腔或非口腔黑色素细菌的全细胞反应。经酶联免疫吸附试验检测，成人牙周炎患者的牙菌斑标本中有一定的免疫反应，而正常人的牙菌斑标本中未检测到免疫反应。患者血清中41K-菌毛抗体和72K-CSP抗体滴度均较高，或分别针对两种蛋白抗原。成人牙周炎晚期41K菌毛特异性Ig G亚类反应升高，以Ig G4为主。该纸片点法简便、快速，可用于P.G.以及检测P.G.特异性抗体。

Porphyromonas gingivalis is an anaerobic gram-negative rod which has been isolated frequently from the periodontal pockets of patients with periodontal disease. The organism has been implicated as a major pathogen in the development of adult periodontitis. The ELISA testing method with a cellulose acetate paper point for detecting P.gingivalis (P.g.) and its antibody responses in subgingival plaque samples were investigated. Using multipin peptide synthesis technology, several sequential overlapping peptides were synthesized. Six and seven immunodominant regions within 41K-fimbriae and 72-CSK protein molecule, which definitely reacted with the serum of patients with periodontal diseases, were detected, respectively. Rabbit polyclonal IgG (Poly Pgf-I) and mouse monoclonal antibodies (mAbs Pgf-I) against 41K-fimbriae were obtained. Poly Pgf-I and mAbs Pgf-I reacted with cell suspensions consisting of the range of 20,000 to 2,000,000 P.g.cells by the ELISA method. The developed paper point could be classified into four degrees -, +, ++ and +++ according to the number of P.g.cells. Poly Pgf-I and mAbs Pgf-I reacted with P.g.cells, while neither of these antibodies reacted with whole cells of other oral or nonoral black-pigmented bacterial species. Analysis by the ELISA method resulted in definite immunoreactivity in subgingival plaque samples of patients with adult periodontitis, while no detectable reactivity was found in those of normal healthy subjects. Among the patients, high titers of both 41K-fimbria-and 72K-CSP-antibodies or against each protein antigens separately were observed in the serum specimens tested. The 41K-fimbriae-specific IgG subclass responses were elevated in the advanced stage of adult periodontitis, and IgG4 was dominant. The simple and rapid ELISA method using the paper point may be useful for the identification of P.g. and detection of P.g.-specific antibodies.