抗幽门螺杆菌尿素酶B亚单位单克隆抗体制备和鉴定及其应用  

Preparation,identification and application of monoclonal antibody against urease subuint B of Helicobacter pylori

作　　者：丁焕弟[1] 韩莹[2] 黄安根 孟圆[1] 胡华新[1] 李晓彤[1] 

机构地区：[1]厦门大学生命科学学院,杂交瘤和抗体技术中心,福建厦门361005 [2]厦门大学附属第一医院检验科,福建厦门361005

出　　处：《细胞与分子免疫学杂志》2014年第12期1278-1281,1286,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：福建省科技重点项目(2011Y0050);厦门市科技计划项目(3502Z20123009);国家基础科学人才培养基金(J1310027)

摘　　要：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 and characterize the monoclonal antibody（mAb） against Helicobacter pylori（Hp）and establish a competitive ELISA used for detection of Hp antibodies in the sera of Hp-infected patients. Methods BALB /c mice were immunized with inactivated Hp to generate Hp mAb using the hybridoma technology. Hp mixed proteins including cytotoxin-associated gene A（CagA）,vacuolating cytotoxin A（VacA） and urease,as well as inactivated Hp were applied to screen positive hybridoma. Selected Hp mAb was analyzed and characterized with ELISA and Western blotting,and then labeled with horseradish peroxidase（ HRP） for establishing a competitive ELISA to detect Hp antibodies in the sera of Hp-infected patients. Results One Hp mAb named as C3 was selected after screening large amount of hybridoma,and the C3 Hp mAb was special for IgG2 a subtype with the affinity titer of 1 × 107. Western blotting,ELISA and mass spectrum analysis indicated that the C3 Hp mAb could recognize Hp urease subunit B specifically. Using the C3 Hp mAb,we developed a competitive ELISA which could be used to detect Hp antibodies in the sera of Hp-infected patients. Conclusion We successfully obtained one mAb that could specifically recognize Hp urease subunit B and developed a competitive ELISA using the Hp mAb.

关 键 词：幽门螺杆菌 单克隆抗体 竞争ELISA 

分 类 号：R392.11[医药卫生—免疫学] R446.61[医药卫生—基础医学]