结核分枝杆菌IFN-γ酶联免疫斑点检测的建立和初步应用

Development of Elispot assay for diagnosis of pulmonary tuberculosis

目的建立结核分枝杆菌特异性IFN-γ酶联免疫斑点(Elispot)检测技术,初步评价其在诊断结核分枝杆菌感染的敏感性和特异性。方法采用以早期分泌抗原靶6 kDa蛋白(ESAT-6)抗原为主的重组蛋白库、多肽库为抗原,建立结核分枝杆菌特异性IFN-γElispot检测技术(简称Elis-pot);应用该技术对32例肺结核病患者、205例健康对照者和18例肺部其他疾病对照的外周血结核菌特异性IFN-γ水平进行检测;结核病人和部分健康对照同时采用全血干扰素试剂(Quantiferon-TB-Gold,QFT-G)进行平行检测。结果采用Elispot检测,结核患者的阳性率最高(75.0%),显著高于健康对照(7.3%)和其他肺部疾病患者(11.1%),结核患者反应水平与其他两组比较均差异有统计学意义(P<0.05,P<0.05)。采用QFT-G在结核病人中的IFN-γ应答阳性率为78.1%,与Elis-pot检测结果进行配对比较无显著性差别(χ2=1.6,P>0.05)。结论建立了结核菌特异性IFN-γElispot检测技术,该技术在诊断结核菌感染的初步应用显示出较好的特异性和敏感性,但仍需进一步扩大样本数观察。

Objective To develop Elispot assays for detection of Mycobacterium tuberculosis anti- gen-specific IFN-γ in peripheral blood and evaluate its applicability in diagnosis of pulmonary tuberculosis. Methods Recombinant protein library containing the early secretary antigen target 6 kDa protein （ESAT-6） and peptide library were used as antigens to develop an Elispot assay for measuring antigen-specific IFN-γ. IFN-γresponses in peripheral bloods were determined in 32 patients with pulmonary tuberculosis（TB）, 205 healthy controls （HC） and 18 patients with lung diseases other than tuberculosis （disease control, DC）. For comparison, 32 patients with TB and 20 healthy controis were also tested with the whole-blood IFN-γ assay（Quantiferon-TB-Gold,QFT-G）in parallel. Results The proportion of positive IFN-γresponse using our in-house Elispot in TB, HC, and DC is 75.0%, 7.3% and 11.1%, respectively. The parallel study using QFT-G showed that 78.1% of TB was positive. There is no significant difference between the results got by our in-house Elispot and QFT-G（χ^2 = 1.6,P〉0.05）. Conclusions An Elispot assay for detection of MTB antigen-specific IFN-γ was developed with higher sensitivity and specificity. The Elispot assay can be applied to diagnosis of TB infection.