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

从腐蚀艾肯氏菌细胞中纯化出一种细菌凝集素样物质（LS），该物质被认为参与了腐蚀艾肯氏菌对多种宿主细胞的粘附。LS在1% Triton X-100中超声提取，经半乳糖胺亲和层析和凝胶过滤层析部分纯化。经特异性血凝活性评价，LS纯化约256倍。研究了一种制备抗LS单克隆抗体（moAnti-LS）的方法。纯化的LS和骨髓瘤细胞SP-2免疫BALB/c小鼠后，将其脾细胞融合，ELISA法筛选杂交瘤。选取4个杂交瘤无性系。其中2例为IgG1同型，其余为IgG2b同型。免疫电镜观察发现，LS可定位于大肠杆菌的细胞壁或荚膜，腐蚀细胞。在共检测的42株口腔细菌中，发现E.锈菌1073与8株共聚集，对E.锈菌1073与粘胶放线菌ATCC19246、a .粘胶菌T14AV、血链球菌34、血链球菌ST160R共聚集反应机制的研究表明，E.锈菌1073与粘胶放线菌ATCC19246、粘胶放线菌T14AV、血链球菌ST160R共聚集反应是由E.锈菌上的LS与伴侣体上的糖受体之间的细胞表面特异性相互作用系统介导的。moAnti-LS能完全抑制锈菌与2株粘链球菌和2株血链球菌的共聚集。moAnti-LS对E.锈菌的血凝也有抑制作用。这些结果表明，moAnti-LS可能特异性地抑制了腐蚀菌在实验动物口腔中的定植。事实上，E. corrosion 1073在SPF大鼠牙周袋内的建立和对牙槽骨的吸附都被n -乙酰- d -半乳糖胺（一种LS的特异性抑制剂）所抑制。

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.