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

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.