结核分枝杆菌抗体双抗原夹心ELISA检测方法的建立及初步应用  

Establishment of Double Antigen Sandwich ELISA and Their Application in Detecting Antibodies for Mycobacterium tuberculosis

作　　者：蒋纾芳 郭兰芹 宗克亮 杨锡琴[1] 刘志强[1] 赵照[5] 修冰水[1] 段翠密[1] 陈堃[1] 张旭辉[1] 张嵘[2] 冯晓燕[1] 张贺秋[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]沈阳药科大学,辽宁沈阳110016 [3]华北石油总医院,河北任丘062552 [4]风帆集团职工医院,河北保定071000 [5]首都师范大学数学科学学院,北京100148

出　　处：《生物技术通讯》2015年第5期678-681,共4页Letters in Biotechnology

基　　金："十二五"国家科技重大专项(2013ZX10004803);"十二五"国家科技重大传染病专项(2013ZX10004-101-003)

摘　　要：目的:建立2种结核分枝杆菌(Mtb)抗体的双抗原夹心ELISA检测方法,并初步评价其在Mtb血清学临床辅助诊断中的应用价值。方法:采用Mtb融合抗原38k D+ESAT6+CFP10作为包被抗原,分别以辣根过氧化物酶(HRP)和生物素(Bio)标记的38k D+ESAT6+CFP10作为标记抗原,建立2种Mtb双抗原夹心ELISA法,即HRP-ELISA法和Bio-ELISA法;采用所建立的2种方法对结核患者和健康对照血清进行检测。结果:经过一系列的反应条件优化,确定HRP-ELISA法和Bio-ELISA法的最佳抗原包被浓度分别为2、0.25μg/m L,最适加样量均为100μL血清原液,最佳标记抗原稀释率分别为1∶500、1∶2000,检测的灵敏度分别为43.66%、52.11%,特异性均为100%。结论:建立了2种Mtb双抗原夹心ELISA法,它们均适用于Mtb血清学的临床辅助诊断。Objective： To set up two kinds of double antigen sandwich ELISA for Mycobacterium tuberculosis （Mtb） antibody, and evaluate the clinical diagnostic significance in Mtb serology preliminarily. Methods： Two Mtb double antigen sandwich ELISA were set up using Mtb polyprotein 38kD＋ESAT6＋CFP10 as coating antigen and HRP or biotin labeled 38kD＋ESAT6＋CFP10 as labeled antigens respectively, namely HRP-ELISA and Bio-ELISA. The antibody levels in serum of tuberculosis patients and healthy individuals were detected using such two double antigen sandwich ELISA methods. Results： Through optimization, the optimum concentration of HRP-ELISA coat- ing antigen and Bio-ELISA coating antigen are 2 and 0.25 μg/mL respectively, the optimum volume of sample are 100 μL, the optimum dilution ratio of labeled antigens are 1：500 and 1：2000. The detection sensitivities are 43.66% and 52.11%, and the specificities are 100%. Conclusion： The two established Mtb double antigen sand- wich ELISA are all suitable for clinical assistant serodiagnosis in Mtb.

关 键 词：结核分枝杆菌 融合抗原38k D+ESAT6+CFP10 双抗原夹心ELISA 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]