|Budget Amount *help
¥5,800,000 (Direct Cost : ¥5,800,000)
Fiscal Year 1996 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1995 : ¥3,400,000 (Direct Cost : ¥3,400,000)
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 poin
t 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.
2)防水加工したペ-パ-ポイントを作成し,所定数のP. gingivalis菌体を同ペ-パ-ポイント上に吸着し,作出したウサギ抗血清やマウスモノクローナル抗体を用いて,P. gingivalisの細菌数とELISAによる反応性において明確な用量-反応関係が得られた.
3)歯周病患者の歯肉溝液,唾液ならびに血清中のP. gingivalis線毛蛋白抗原やそのエピトープに対応するペプチド抗原に対する特異抗体を調べ,その反応性と歯周病との関係を検討した.その結果,P. gingivalisの2つのタイプの線毛蛋白抗原やそのB細胞エピトープのペプチドと患者歯肉溝液および同血清と明確な反応がみられた.また,2つのタイプの線毛蛋白抗原に両方反応する血清やそれぞれの線毛抗原にしか反応しない血清が認められた.さらに,歯肉溝液との反応において調べた限りでは,特にIgGサブクラスにおいて病態の悪化にともないIgG4サブクラスの反応性が強まる傾向が見られた. Less