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

测定了放线菌血清型特异性多糖抗原的化学结构。采用高压灭菌法从放线菌全细胞中提取粗抗原。通过离子交换层析和凝胶过滤对提取物进行纯化。血清a、b和c型抗原分别由重复单位-3)-6-deoxy- α -d - tal_p -(1-2)-6-deoxy- α -d - tal_p -(1-, -3)- α -d - fuc_p -(1-2)- α - l - rha_p -(1-和-3)-6-deoxy- α - l - tal_p -(1-)-6-deoxy- α - l - tal_p -(1-)组成。制备了21个能产生抗放线菌单胞菌Y4全细胞单克隆抗体的杂交瘤。其中，8个单抗（单克隆抗体Sl-S8）与血清型b抗原反应，3个单抗（单克隆抗体Ll-L3）与菌株Y4的脂多糖反应，4个单抗（单克隆抗体Pi-P4）与菌株Y4的细胞表面蛋白抗原反应。用这些单克隆抗体致敏的乳胶颗粒被允许与整个细胞和牙周细菌的细胞提取物反应。用MAb L2致敏的乳胶颗粒与所有三种血清型放线菌的全细胞和培养上清凝固。另一方面，MAb S5致敏乳胶颗粒对血清型b a放线菌comitans的全细胞、培养上清液和牙龈拟杆菌（Bacteroides gingivalis）全细胞均有阳性凝集，而对加热（100 C, 3 min）的牙龈拟杆菌（Bacteroides gingivalis）细胞无阳性凝集。单克隆抗体的快速乳胶凝集试验可用于放线菌的鉴定和血清分型。

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.