结核抗原时间分辨荧光免疫分析的建立及其初步临床应用

Establishment of time-resolved fluoroimmunoassay for mycobacterium tuberculosis antigen

目的：建立时间分辨荧光免疫分析法（TRFIA）检测结核分枝杆菌特异性6000早期分泌性抗原靶（ESAT-6）和抗原培养滤液蛋白10（CFP10）的融合抗原，并对该方法诊断肺结核的性能进行评价。方法采用夹心法建立ESAT-6-CFP10的TRFIA法。并对其灵敏度、批内、批间差异和准确性等各项指标进行考核。试验组选取24例活动性、32例可疑活动性、65例非活动性结核病患者及419例非结核性肺部疾病患者；30名健康体检者作为对照组，比较TRFIA法与酶联免疫吸附试验（ELISA）法检测结果。进行临床检测并与临床诊断进行一致性检验分析。结果 ESAT-6/CFP10的TRIFA法稳定性好，线性宽，其检测灵敏度为2.5 ng/mL，批内和批间变异系数分别平均为3.37%和5.23%，平均回收率为97.5%。与商业ELISA试剂盒一致性较好（Kappa=0.914，P=0.01）。 TRFIA法的剂量-反应曲线拟合相关系数（R2）达0.9961，可测范围为（2.5-1600） ng/mL；该方法与临床诊断有较好的一致性（Kappa=0.961，P=0.001）。结论 TRFIA法敏感度、精密度、重复性、线性相关性较好，有利于该结核抗原用于临床结核病的诊断。

Objective To establish a time-resolved fluorimmunoassay （TRFIA） for detecting the ESAT-6/CFP10 antigen and evaluate its clinic application. Methods A sandwich TRFIA of ESAT-6/CFP10 was established. The samples of ESAT-6/CFP10 in 24 patients with active tuberculosis, 32 patients with suspected active tuberculosis, 65 patients with inactivity tuberculosis, 419 non-tuberculous patients and 30 normal controls were measured. The sensitivity, precision and repetition of TRFIA were detected. The value of ESAT-6/CFP10 in the diagnosis mycobacterium tuberculosis infection were evaluated by using the consistency check of diagnostic test. Results The correlation coefficient （r2） of TRFIA dose-response curve reached 0.996 1. The detectability of detecting ESAT-6/CFP10 was 2.5 ng/mL, and the measurement range was （2.5-1 600） ng/mL. The inter and intra assay CVs were 3.37% and 5.23%, respectively. The recovery rate was 97.5%. TRFIA had high-level consistency with ELISA and clinical diagnosis（Kappa=0.914, P=0.001;Kappa=0.961, P=0.001; respectively）. Conclusion The established sandwich TRFIA for ESAT-6/CFP10 measurement is a practicable assay. The results of precision, repetition, linear correlation and preliminary clinic application are satisfactory. It is helpful to diagnose mycobacterium tuberculosis infection with detecting ESAT-6/CFP10.