免疫磁性微球富集检测结核分枝杆菌方法的建立  

Establishment of a method for separation of Mycobacterium tuberculosis by immunomagnetic beads

作　　者：古丽仙.吐尔逊 顾志冬[1] 陈长强[1] 周敏[2] 卫蓓文[3] 张海晨[4] 费凤英[5] 樊绮诗[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院检验科,上海200025 [2]上海交通大学医学院附属瑞金医院微生物科,上海200025 [3]上海交通大学医学院附属瑞金医院医学检验系,上海200025 [4]上海市徐汇区中心医院检验科,上海200031 [5]上海复旦大学附属青浦中心医院检验科,上海201700

出　　处：《诊断学理论与实践》2012年第5期476-480,共5页Journal of Diagnostics Concepts & Practice

基　　金：上海市科委(10411965800);上海申康医院发展中心(SHDC12010215)

摘　　要：目的:建立一种基于免疫磁性微球富集的检测结核分枝杆菌的新方法 ,以提高其临床阳性检出率。方法:提取结核分枝杆菌H37Rv抗原,并免疫新西兰白兔,制备兔抗结核分枝杆菌H37Rv血清。分离纯化抗血清,采用酶联免疫吸附试验和SDS-聚丙烯酰胺凝胶电泳技术鉴定抗体的效价和纯度。将抗结核分枝杆菌抗体与生物素标记,并与结合有链霉亲和素的磁性微球进行连接获得免疫磁性微球,优化反应条件,建立免疫磁性微球富集检测结核分枝杆菌的方法。以培养结果作为判断金标准,比较免疫磁性微球富集方法与常规涂片镜检方法的检出率。结果:制备的抗结核分枝杆菌抗体浓度为14 mg/mL,最终效价为1∶20 000。检测158例临床标本,免疫磁性微球富集方法可使涂片镜检阳性率从5.1%提高到13.9%。22例免疫磁性微球富集后涂片镜检阳性的标本培养均为阳性,其特异度为100%。结论:建立的免疫磁性微球富集方法简便、有效,能显著提高结核分枝杆菌镜检的灵敏度和特异度。Objective： To establish a new method for Mycobacterium tuberculosis detection by using immunomagnetic beads technology. Methods： Immunizing New Zealand rabbits with H37Rv antigen extracted from Mycobacterium tuberculosis and thereafter the rabbit anti-H37Rv antiserum was prepared. A highly purified antiserum of anti-Mycobacterium tuberculosis antibody was obtained. Titer and purity of anti-Mycobacterium tuberculosis antibody were identified using ELISA and SDS-PAGE technology. Anti-Mycobacterium tuberculosis antibodies were labeled by biotin and connected with magnetic beads which were marked by streptavidin. After optimizing the reaction condition, the method of immunomagnetic beads separation （IMS） for detection of Mycobacterium tuberculosis was established. The detection rates of Mycobacterium tuberculosis by IMS-smear microscopy and routine direct smear microscopy were compared using the result of culture as the golden standard. Results： Concentration of anti-Mycobacterium tuberculosis antibody was 14 mg/ mL, and it could be diluted to a maximum of 1：20 000. Mycobacterium tuberculosis was detected in 158 clinical specimens using immunomagnetic beads separation. The positive rate was increased from 5.1% in direct smear to 13.9% in IMS-smear; the 22 positive specimens detected by immunomagenetic beads separation were also positive by culture, the specificity was 100%. Conclusions： Immunomagnetic beads separation is a convenient and effective method, it could improve the sensitivity and specificity of smear microscopy and be recommended for clinical application.

关 键 词：结核分枝杆菌 免疫磁性微球 磁性分离富集 涂片镜检 培养 

分 类 号：R521[医药卫生—内科学]