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幽门螺杆菌(Helicobacter pylori,Hp)是一种定居于人胃部及十二指肠的微需氧革兰氏阴性菌,其长期感染导致胃炎、胃及十二指肠溃疡与胃癌。幽门螺杆菌感染主要始于儿童期,其自发性清除非常少见,及早通过检测发现并加以抗生素治疗可有效降低成人感染率。本文通过体外培养Hp标准菌株ATCC43504,将其裂解液作为抗原免疫Balb/c小鼠,通过常规技术路线制备杂交瘤细胞。经间接ELISA法筛选和多次有限稀释法亚克隆得到21株稳定分泌抗Hp抗原的杂交瘤细胞株,制备腹水并用辛酸硫酸铵联合沉淀法纯化单克隆抗体。将得到的单克隆抗体在胶体金免疫层析平台上进行配对和样品检测,获得多组灵敏度和特异性相对较高的单克隆抗体组合,其中以2-18E1作为包被抗体,2-9H5或2-17E9作为标记抗体检测细菌培养裂解物的最低限为25 ng/m L,为Hp抗原检测试剂的研发提供了基础。
Helicobacter pylori (Hp) is a micro-aerobic gram-negative bacterium that colonizes the human stomach and the duodenum. Its long-term infection leads to gastritis, gastric and duodenal ulcers and gastric cancer. Helicobacter pylori infection mainly begins in childhood, and its spontaneous clearance is very rare. Early detection and antibiotic treatment can effectively reduce the rate of adult infection. In this paper, Hp standard strain ATCC43504 was cultured in vitro, and its lysate was used as an antigen to immunize Balb / c mice. The hybridoma cells were prepared by routine techniques. Twenty-one hybridoma cell lines stably secreting anti-Hp antigens were obtained by indirect ELISA and multiple limiting dilution methods. Ascites was prepared and the monoclonal antibodies were purified by ammonium caprylate-ammonium sulfate precipitation. The obtained monoclonal antibodies were paired with samples on a gold immunochromatography platform to obtain a plurality of monoclonal antibody combinations with relatively high sensitivity and specificity, in which 2-18E1 was used as coating antibody, 2-9H5 or The minimum limit of 2-17E9 as a labeled antibody for the detection of bacterial culture lysates is 25 ng / mL, which provides the basis for the development of Hp antigen detection reagents.