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性革兰氏阴性杆状菌，经常从牙周病患者的牙周袋中分离出来。该有机体已被认为是成人牙周炎发展的主要病原体。研究了醋酸纤维素纸点ELISA法检测牙龈假单胞菌（P.g）及其在龈下菌斑中的抗体反应。采用多针多肽合成技术，合成了多个顺序重叠的多肽。在41k -菌膜和72-CSK蛋白分子中分别检测到6个和7个免疫优势区，这些免疫优势区与牙周病患者的血清有明确的反应。获得兔多克隆IgG （Poly Pgf-I）和小鼠单克隆抗体（maabs Pgf-I）。聚Pgf-I和单克隆抗体Pgf-I通过ELISA方法与包含20,000至2,000,000个p.g.g细胞的细胞悬液反应。根据p.g.g cell的数量，可将开发的纸点分为-、+、++和+++四个度。Poly - Pgf-I和mab - Pgf-I与p.g.s细胞反应，而这两种抗体都不能与其他口服或非口服黑色色素细菌的整个细胞反应。ELISA法分析发现，成人牙周炎患者的龈下菌斑样本存在明确的免疫反应性，而正常健康受试者的龈下菌斑样本未发现可检测到的免疫反应性。在患者中，检测的血清标本中分别观察到41k - fibria和72k - csp抗体或针对每种蛋白抗原的高滴度。在成人牙周炎晚期，41k -菌毛特异性IgG亚类反应升高，IgG4占主导地位。该方法简便、快速，可用于p.g.g的鉴定和p.g.g特异性抗体的检测。

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.