抗结核分枝杆菌早期分泌性抗原靶蛋白6的新型单克隆抗体的制备及其临床应用  

Preparation and clinical application of a novel monoclonal antibody against Mycobacterium tuberculosis early secretory antigenic target-6

作　　者：游璇[1] 冷建杭[1] 寿成珉[2] 吴志刚[3] 沈俊娅[1] 黄晟[4] YOU Xuan LENG Jian - hang SHOU Cheng - min WU Zhi - gang SHEN Jun - ya HUANG Sheng(Central Laboratory, the First People＇s Hospital of Hangzhou Affiliated to Zhefiang University of Chinese Tranditonal Medicine, Hangzhou, Zhejiang 310006, China)

机构地区：[1]浙江省中医药大学附属杭州市第一人民医院中心实验室,浙江杭州310006 [2]杭州市红十字会医院内分泌科,浙江杭州310003 [3]浙江大学医学院附属第一医院传染病诊治国家重点实验室,浙江杭州310003 [4]浙江省中医药大学附属杭州市第一人民医院呼吸科,浙江杭州310006

出　　处：《中国卫生检验杂志》2017年第5期609-611,615,共4页Chinese Journal of Health Laboratory Technology

基　　金：国家"十二.五"传染病防治重大专项课题(2012ZX1-0001-003);杭州市科技发展计划项目(20100633B01)

摘　　要：目的制备抗结核分枝杆菌早期分泌性抗原靶蛋白6(ESAT-6)单克隆抗体,以应用于多种检测技术和临床研究。方法设计并合成结核分枝杆菌ESAT-6特定抗原肽序列,免疫BALB/c小鼠,取其脾细胞和小鼠骨髓瘤细胞SP2/0融合,经酶联免疫吸附试验(ELISA)检测、多次有限稀释亚克隆建立能稳定分泌抗结核分枝杆菌特异抗原肽单克隆抗体的杂交瘤细胞系,纯化后行免疫球蛋白亚型分析,并鉴定分析该单克隆抗体在蛋白质免疫印迹法、免疫沉淀法和酶联免疫法(ELISA)检测中的应用效果。收集结核分枝杆菌培养上清液样本38份,非结核分枝杆菌培养上清液样本20份,结核性胸水样本32份,非结核性胸水样本24份以及健康对照血清样本20份。结果单克隆抗体免疫球蛋白亚型分析显示抗体类型为IgM、κ型,并可应用于Western blotting和免疫沉淀法。ELISA定量检测显示该特异性抗体检测结核分枝杆菌的敏感度为95.7%(67/70),特异度为100%(64/64)。结论 ESAT-6单克隆抗体用于结核分枝杆菌检测有较高的敏感性和特异性,可用于结核病的早期诊断。Objective To prepare a novel monoclonal antibody that specifically against Mycobacterium tuberculosis early secretory antigenic target-6（ ESAT-6）.Methods BALB/c mice were immunized by prepared peptide of ESAT-6,and then the splenocytes of mice were fused with myeloma cell line SP2/0.The resultant fused cells were subjected to screening culture,enzyme linked immunosorbent assay（ ELISA） assay and subcloning by limited dilution to establish hybridoma cell lines of stable secreting anti-the peptide of ESAT-6 antibody.The antibody was purified,and its isotypes were analyzed.Then,the antibody was further evaluated by Western blotting,immunoprecipitation and ELISA in 38 culture supernatant samples of Mycobacterium tuberculosis,20 culture supernatant samples of non-Mycobacterium tuberculosis,32 samples of tuberculous pleural effusion,24 samples of non-tuberculous pleural effusion,and 20 serum samples from healthy controls.Results The types of antibody against the specific peptide of ESAT-6 were IgM and κ type,and it was applicable for Western blotting and immunoprecipitation analysis.ELISA quantitative test showed that the sensitivity and specificity for diagnosis of Mycobacterium tuberculosis were 95.7%（ 67/70） and 100%（ 64/64）,respectively.Conclusion The antibody generated against the specific peptide of ESAT-6 is high in sensitivity and specificity,and it can be used in the early diagnosis of tuberculosis.

关 键 词：结核分枝杆菌 早期分泌性抗原靶蛋白6 抗原肽 单克隆抗体 

分 类 号：R392[医药卫生—免疫学]