Hijiya Takahiro, Shibata Yasuko, Hayakawa Mitsuo, Abiko Yoshimitsu
Department of Biochemistry and Molecular Biology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
Hybridoma (Larchmt). 2010 Jun;29(3):201-4. doi: 10.1089/hyb.2009.0109.
The periodontal pathogen Porphyromans gingivalis is classified into six groups (types I-V and Ib) based on the genotype of the fimbriae A (fimA) gene. Among genotypes, fimA type II strains are thought to be most strongly related to advanced periodontitis. The present study was undertaken to develop passive immunotherapy monoclonal antibodies (MAbs) against periodontitis, which are capable of inhibiting virulency and were constructed through the immunization of outer membrane vesicles (OMV) fraction of fimAII strain, TDC60, using mouse hybridoma technology. MAbs that recognized OMV by ELISA assay were identified, and 28 clones were screened by Western blot analysis. After purifying these MAbs using protein G column, the effect of the MAb on IL-8 production from human gingival fibroblasts by OMV was examined. We selected MAb TDC4-33H, which strongly inhibited the IL-8 production with a higher MAb production rate. Since the MAb showed an individual ladder-like profile against OMV by Western blotting, we further examined the reactivity against lipopolysaccharides (LPS) from TDC60, W83 (fimAIV), and ATCC33277 (fimAI). As a result, MAb TDC4-33H recognized all LPSs. Moreover, MAb TDC4-33H significantly inhibited the LPS-stimulated IL-8 production in human gingival fibroblasts. These findings suggest that MAb TDC4-33H reacts with LPS and may be useful for passive immunotherapy through neutralizing IL-8 production in gingival fibroblasts by P. gingivalis LPS.
牙周病原体牙龈卟啉单胞菌根据菌毛A（fimA）基因的基因型分为六组（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关系最为密切。本研究旨在开发针对牙周炎的被动免疫治疗单克隆抗体（MAb），这些抗体能够抑制毒力，并通过小鼠杂交瘤技术，利用fimA II型菌株TDC60的外膜囊泡（OMV）组分进行免疫构建而成。通过ELISA检测识别OMV的单克隆抗体被鉴定出来，并通过蛋白质印迹分析筛选出28个克隆。使用蛋白G柱纯化这些单克隆抗体后，检测了该单克隆抗体对OMV诱导人牙龈成纤维细胞产生白细胞介素-8的影响。我们选择了单克隆抗体TDC4-33H，它能强烈抑制白细胞介素-8的产生，且单克隆抗体产生率较高。由于该单克隆抗体在蛋白质印迹中对OMV呈现出独特的阶梯状图谱，我们进一步检测了它对TDC60、W83（fimA IV型）和ATCC33277（fimA I型）的脂多糖（LPS）的反应性。结果，单克隆抗体TDC4-33H识别所有的脂多糖。此外，单克隆抗体TDC4-33H显著抑制脂多糖刺激人牙龈成纤维细胞产生白细胞介素-8。这些发现表明，单克隆抗体TDC4-33H与脂多糖反应，可能通过中和牙龈卟啉单胞菌脂多糖诱导牙龈成纤维细胞产生白细胞介素-8而用于被动免疫治疗。
