幽门螺杆菌单克隆抗体的制备及其检测应用

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。

Development of Monoclonal Antibodies against Helicobacter pylori and Their Applications to Immunological Assays

Helicobacter pylori (Hp) is a gram-negative, microaerophilic bacterium found in the human stomach and duodenum, and long-term H. pylori infection is linked to the development of gastritis, gastric and duodenal ulcers, and stomach cancer. H. pylori infection is mainly acquired during childhood, and its spontaneous clearance is rare. Its prevalence among adults can be effectively reduced by early diagnosis and antibiotic therapy. In this study, a standard H. pylori strain (ATCC 43504) was cultured in vitro, the lysate was used as an antigen to immunize BALB/c mice, and a hybridoma cell line was prepared using conventional techniques. In total, 21 hybridoma cell lines that stably secreted anti-H. pylori monoclonal antibodies (mAbs) were obtained based on indirect enzyme-linked immunosorbent assay (ELISA) screening and multiple rounds of limiting dilution subcloning. Ascites were prepared and mAbs were purified using ammonium sulfate precipitation methods. The pairing of mAbs and sample detection were conducted by colloidal gold immunochromatographic assays (GICAs), and multiple mAb pairs with high sensitivity and specificity were obtained. Among them, the limits of detection (LODs) of the lysates from two antibody pairs consisting of capture (mAb 2-18E1) and detection (mAb 2-9H5 and mAb 2-17E9) antibodies were 25 ng/mL. These results indicated that the GICA method can provide a basis for the development of reagents to detect H. pylori antigens.