Development of serotyping reagents for the periodontopathogenic bacterium, Bacteroides (Porphyromonas) gingivalis.

牙龈拟杆菌被认为是与成人牙周炎有关的最致命的细菌之一。该菌的血清学特征是至少有四种不同生物活性的类型，如胶原酶和蛋白水解酶以及抗菌草。本研究的目的是通过产生针对这四种血清型牙周炎乳杆菌的特异性抗原的单抗来建立分型试剂。在弗氏不完全佐剂中，用血清型代表性菌株(I型381株、II型JH4株、III型22KNB-12株和IV型16NH2-1株)免疫BALB/c小鼠。免疫小鼠脾细胞与P3X6-Ag8融合。U1(P3U1)髓鞘细胞在IMDM中使用45%的聚乙二醇组和10%的二甲基亚砜。通过HAT选择选择杂交细胞后，用ELISA筛选产生仅与用于免疫的菌株反应的NaB的克隆。在杂交瘤…产生的单抗中与菌株381反应8株，与JH4反应22株，与22KN6-12反应3株，与16NH2-1反应8株。与381株单抗反应的8株单抗中，有5株与其他I型株包括参考株和新分离株均呈阳性反应，与其他血清型群株无交叉反应。同样，在抗JH4的22个单抗中有4个和抗16NH2-1的8个单抗中有1个与属于其相应血清型组的其他菌株发生反应。其中一些单抗是热不稳定的，另一些是热稳定的，这表明抗原决定簇由多肽或多糖组成。4个血清型菌株提取液的SDS-PAGE图谱基本一致，但用免疫印迹法检测到II、IV型单抗分别与同一血清型菌株50 KDa和100 KDa的抗原反应，提示这些区带可能是一种血清型特异性抗原。本研究获得的单抗可用于成人牙周炎患者感染特征的分型。较少

Bacteroides (Porphyrosonas) gingivilis has been considered one of the most virulent bacteria in relation to adult periodontitis. This bacterium has been serologically characterized to have at least four types with different biological activities such as collagenolytic and proteolytic enzymes and antibiograss. The purpose of this study is to develop the serotyping reagents by producing monoclonal antibodies (MAbs) for specific antigens of these four serotypes of lacteroides gingivalis.BALB/c mice were intraperitoneally immunized with serotype representative strains (381 for serotype I, JH4 for serotype II, 22KNB-12 for serotype IIII nd 16NH2-1 for serotype IV) in Freund incomplete adjuvant. Spleen cells from immunized mouse were fused with P3X6-Ag8. U1 (P3U1) myelona cells usins 45% PEG, 10% DMSO in IMDM. After selecting hybrid cells by HAT selection, clones producing NAbs that reacted only with a strain used for the immunization were screened by ELISA.Among MAbs produced by hybridomas … More which developed a positive reaction in ELISA, 8 MAbs reacted with the strain 381, 22 with JH4, 3 with 22KN6-12 and 8 with 16NH2-1, respevtively. Five out of 8 MAbs, which reacted with a strain 381, developed a positive reaction also with other serotype I strains including reference and freshly isolated ones, without cross-reacting with other serotype group strains. Similarly, 4 out of 22 MAbs against JH4 and 1 out of 8 MAbs against 16NH2-1 reacted with other strains belonging to their corresponding serotype groups. Some of these MAbs were heat-labile and others were heat-stable, suggesting that antigenic determinants consist of either peptide or polysaccharide. SDS-PAGE patterns of bacterial extract from 4 serotype groups were almost similar, but when the MAb was reacted with heat-treated (heat-stable) antigens by a Western blotting procedure, MAb asainst strains from serotype II and IV reacted with an antigen of 50 and 100 KDa from the same serotype strain, respectively, suggesting that these bands could be an serotype specific antigens. MAbs obtained in this study might be useful in classifying infectious features of the patients with adult periodontitis. Less