Studies on colonization inhibition of periodontopathic bacteria -Utilization of the monoclonal antibodies-

从腐蚀性艾美耳球虫细胞中分离纯化出一种细菌凝集素样物(LS)，该物质被认为参与了Eikenella腐蚀物与多种宿主细胞的黏附。用1%Triton X-100超声法从腐蚀菌1073的细胞膜中提取LS，并用氨基半乳糖亲和层析和凝胶过滤层析进行部分纯化。用比血凝活性评价LS的纯度，纯化倍数约为256倍。将纯化的LS免疫BALB/c小鼠，取脾细胞与骨髓瘤细胞(SP-2)融合，用ELISA法筛选杂交瘤细胞。筛选出4个杂交瘤克隆。其中2例为IgG1亚型，1例为IgG2b亚型。免疫电子显微镜研究发现，LS定位于腐蚀性肠杆菌的细胞壁或囊膜中。腐蚀菌1073与42株口腔细菌中的8株发生共聚集，对腐蚀性肠杆菌1073与粘性放线菌ATCC19246、粘性放线菌T14AV、血链球菌34和血链球菌ST160R的共聚集机制的研究表明，腐蚀性肠杆菌上的LS与其伴侣上的糖受体之间的特异性细胞表面相互作用系统介导了共聚集。Moanti-LS对腐蚀性肠杆菌的血凝有抑制作用。这些结果表明，moanti-LS可能特异性地抑制腐蚀性肠杆菌在实验动物口腔中的定植。事实上，LS的一种特异性抑制剂N-乙酰-D-氨基半乳糖可抑制SPF大鼠牙周袋中腐蚀性E.1073的建立和牙槽骨在体内的吸附。

A bacterial lectin-like substance (LS), which is considered to participate in the adherence of Eikenella corrodens to various host cells, was purified from E. corrodens cells. The LS was extracted in 1% Triton X-100 with sonication from the cell envelope of E. corrodens 1073, and partially purified by galactosamine affinity chromatography and gel filtration chromatography. The LS was purified about 256-fold as evaluated by its specific hemagglutinating activity.A method to produce monoclonal antibody against the LS (moAnti-LS) was investigated. Splenocytes of BALB/c mice immunized with purified LS and myeloma cells (SP-2) were fused, and hybridomas were screened by ELISA. Four hybridoma clones were selected. Two of them were of the IgG1 isotype and the others were IgG2b isotype. Immunoelectron microscopic study revealed that LS was found to localize in cell wall or capsule of E. corrodens cells.E. corrodens 1073 was found to coaggregate with 8 strains of total of 42 oral bacterial strains examined, and the study on mechanisms of the coaggregation reactions of E. corrodens 1073 with Actinomyces viscosus ATCC19246, A. viscosus T14AV, Streptococcus sanguis 34, and S. sanguis ST160R indicates that the coaggregations are mediated by a system of specific cell surface interaction between the LS on E. corrodens and sugar receptors on the partner.The moAnti-LS completely inhibited the coaggregation of E. corrodens with 2 strains of A. viscosus and 2 strains of S. sanguis. The moAnti-LS also inhibited hemagglutination of E. corrodens. These results suggested that colonization of E. corrodens in the mouth of experimental animal may be specifically inhibited by the moAnti-LS. Indeed, the establishment of E. corrodens 1073 in the periodontal pocket and the adsorption of the alveolar bone of SPF rats in vivo were both inhibited by administration of N-acetyl-D-galactosamine, a specific inhibitor of the LS.