Maruyama Mitsuhiro, Hayakawa Mitsuo, Zhang Lin, Shibata Yasuko, Abiko Yoshimitsu
Department of Biochemistry and Molecular Biology, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Hybridoma (Larchmt). 2009 Dec;28(6):431-4. doi: 10.1089/hyb.2009.0055.
An important periodontal pathogen, Porphyromans gingivalis strains are classified into six genotypes (types I-V and Ib), based on the genotype of the fimbriae A (fimA). Among the genotypes, fimA type II strains are thought to be most strongly related to advanced periodontitis. To develop passive immunotherapy, over 300 hybridoma clones were constructed through immunization of cell extracts of fimA type II strain P. gingivalis TDC60 using hybridoma technology. Among these clones, 15 MAbs recognized TDC60 lipopolysaccharide (LPS) with an individual ladder-like structure by Western blot analysis. Further Western blotting of the 15 MAbs against LPS from TDC60, FDC381 (fimA type I), and W83 (fimA type IV) of P. gingivalis and Escherichia coli was carried out. None of these MAbs recognized E. coli LPS, and divided into at least three different Western blot patterns. To confirm the specificity to LPS, three clones were selected and competition assays were carried out using TDC60 LPS. All three MAbs reduced the reactivity against TDC60 LPS after absorption of the LPS in a dose-dependent manner. These findings suggest that MAbs recognizing different epitopes of P. gingivalis LPS were successfully constructed, and these MAbs may be useful in neutralizing P. gingivalis infection.
牙龈卟啉单胞菌是一种重要的牙周病原体,根据菌毛A(fimA)的基因型,牙龈卟啉单胞菌菌株可分为六种基因型(I-V型和Ib型)。在这些基因型中,fimA II型菌株被认为与重度牙周炎的关系最为密切。为了开发被动免疫疗法,利用杂交瘤技术,通过免疫fimA II型菌株牙龈卟啉单胞菌TDC60的细胞提取物构建了300多个杂交瘤克隆。在这些克隆中,15种单克隆抗体(MAb)通过蛋白质印迹分析识别出具有单个梯状结构的TDC60脂多糖(LPS)。进一步对这15种MAb针对牙龈卟啉单胞菌的TDC60、FDC381(fimA I型)和W83(fimA IV型)以及大肠杆菌的LPS进行蛋白质印迹分析。这些MAb均未识别出大肠杆菌LPS,且至少分为三种不同的蛋白质印迹模式。为了确认对LPS的特异性,选择了三个克隆,并使用TDC60 LPS进行竞争试验。所有三种MAb在以剂量依赖方式吸收LPS后,均降低了对TDC60 LPS的反应性。这些发现表明,成功构建了识别牙龈卟啉单胞菌LPS不同表位的MAb,这些MAb可能有助于中和牙龈卟啉单胞菌感染。