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目的制备抗幽门螺杆菌(Hp)的单克隆抗体(mAb),并对该Hp mAb进行分析鉴定,建立一种检测患者由于Hp感染而在血清中产生Hp抗体的竞争ELISA检测方法。方法用灭活的Hp免疫BALB/c小鼠,通过杂交瘤技术制备Hp mAb。我们采用Hp混合蛋白包括毒素相关蛋白A(CagA)、空泡毒素A(VacA)和尿素酶以及灭活的Hp菌体筛选阳性杂交瘤细胞株,用ELISA和Western blot等技术对所获得的Hp mAb进行鉴定。利用辣根过氧化物酶(HRP)标记所筛选的Hp mAb来建立一个可检测患者血清中Hp抗体的竞争ELISA。结果通过大规模的杂交瘤筛选,我们选择了1株将其命名为C3 Hp mAb,其抗体亚型为IgG2a,腹水效价可达1×107。Western blot法、ELISA和质谱检测结果显示,该C3 Hp mAb能特异地识别Hp的尿素酶B亚单位。用这个C3 Hp mAb,我们建立了一种可检测患者血清中Hp抗体的竞争ELISA。结论成功获得一种可以特异识别Hp尿素酶B亚单位的mAb,建立了一种可检测患者血清中Hp抗体的竞争ELISA。
OBJECTIVE To prepare a monoclonal antibody (mAb) against Helicobacter pylori (Hp), and to analyze and identify this Hp mAb, a competitive ELISA method for detecting Hp antibodies in serum was developed in patients with Hp infection. Methods BALB / c mice were immunized with inactivated Hp and Hp mAbs were prepared by hybridoma technique. We screened positive hybridoma cell lines using Hp mixed proteins including CagA, VacA and urease, and inactivated Hp mycelia. The obtained Hp mAb for identification. A horse serum peroxidase (HRP) -labeled Hp mAb was used to establish a competitive ELISA that detects Hp antibodies in patient sera. Results We screened a large number of hybridomas and chose a strain named C3 Hp mAb with IgG2a antibody subtype and ascitic fluid titer of 1 × 107. Western blot, ELISA and mass spectrometry showed that the C3 Hp mAb could specifically recognize the urease B subunit of Hp. Using this C3 Hp mAb, we established a competitive ELISA that detects Hp antibodies in patient sera. Conclusion A mAb that can specifically recognize the B subunit of Hp urease was successfully obtained and a competitive ELISA was developed to detect Hp antibodies in serum of patients.