Development of simple and rapid method using monoclonal antibodies for identification of periodontal bacteria

测定了伴生放线杆菌血清型特异性多糖抗原的化学结构。用高压灭菌法从伴生放线菌全细胞中提取粗抗原。提取液经离子交换层析和凝胶过滤纯化。A、b和c型抗原由重复单位-3)-6-deoxy-alpha-D-Tal_p-(1-2)-6-deoxy-alpha-D-Tal_p-(1-，-3)-α-D-FUC_p-(1-2)-α-L-Rha_p-(1-和-3)-6-deoxy-alpha-L-Tal_p-(1-2)-6-deoxy-alpha-L-Tal_p-(1-，)组成分别对21株产生抗放线菌伴生菌Y4株全细胞单抗的杂交瘤细胞株进行了筛选。其中8株单抗(单抗S1-S8)与b型抗原反应，3株单抗(单抗L1-L3)与Y4菌株的脂多糖反应，4株单抗(单抗PI-P4)与Y4株细胞表面蛋白抗原反应。用单抗L2致敏的乳胶颗粒与所有三种血清型伴生放线杆菌的全细胞和培养上清液凝集。另一方面，用单抗S5致敏的胶乳颗粒与放线菌伴生型b A全细胞、培养上清液和牙龈拟杆菌全细胞均能产生阳性凝集，但与加热(100minﾟC，3min)的牙龈杆菌细胞不能产生阳性凝集。

The chemical structures of serotype-specific polysaccharide antigens from Actinobacillus actinomycetemcomitans were determined. Crude antigens were extracted from whole cells of A actinomycetemcomitans by autoclaving. The extracts were purified by ion-exchange chromatography and gel filtration. The serotype a, b and c antigens were composed of repeating units, -3)-6-deoxy-alpha-D-Tal_p-(1-2)-6-deoxy-alpha-D-Tal_p-(1-, -3)-alpha-D-Fuc_p-(1-2)-alpha-L-Rha_p-(1- and -3)-6-deoxy-alpha-L-Tal_p-(1-2)-6-deoxy-alpha-L-Tal_p-(1-, respectively.Twenty-one hybridomas producing monoclonal antibodies (MAbs) to whole cells of A. actinomycetemcomitans strain Y4 were elaborated. Among these MAbs, eight MAbs (MAb Sl-S8) reacted with the serotype b antigen, three MAbs (MAb Ll-L3) reacted with lipopolysaccharide from strain Y4, and four MAbs (MAb Pi-P4) reacted with cell-surface protein antigens of strain Y4.Latex particles sensitized with these MAbs were allowed to react with whole cells and cell extracts of periodontal bacteria. Latex particles sensitized with MAb L2 coagglutinated with whole cells and culture supernatants of all three serotypes of A. actinomycetemcomitans. On the other hand, latex particles sensitized with MAb S5 produced positive agglutination with whole cells and culture supernatants of serotype b A. actinomycetemcomitans and whole cells of Bacteroides gingivalis, but not with heated (100ﾟC, 3 min) cells of B. gingivalis.The simple and rapid latex agglutination assay using MAbs may be useful for the identification and serotyping of A. actinomycetemcomitans strains.