CFP10/ESAT6融合蛋白-ELISPOT方法的建立及其在结核分枝杆菌感染诊断中的应用价值

目的建立应用酶联免疫斑点试验(Enzyme Linked-Immunospot Assay,ELISPOT)检测结核分枝杆菌感染的方法 ,评价CFP10/ESAT6融合蛋白抗原在检测结核分枝杆菌感染中的应用价值。方法通过比较不同的实验条件确定ELIS-POT检测结核分枝杆菌感染方法的最佳细胞浓度、孵育时间、蛋白抗原浓度等,建立ELISPOT方法。以PPD皮肤试验作对照。结果 ELISPOT方法的最佳实验条件为:每孔细胞浓度2×105,CFP10/ESAT6融合蛋白抗原浓度20~40μg/mL,细胞孵育时间20h。应用PPD皮试检测30例健康体检者、38例非结核疾病患者和40例结核病患者的阳性率分别为40.0%、0%、72.5%,而ELISPOT检测的阳性率分别为20.0%、10.5%、92.5%。应用ELISPOT检测40例结核病痰菌阳性组和阴性组的灵敏度分别为94.4%、90.9%。结论应用CFP10/ESAT6融合蛋白作为抗原建立的ELISPOT方法可初步应用于诊断结核分枝杆菌感染、辅助结核病诊断。

CFP10/ESAT6 fusion protein-ELISPOT method for Mycobacterium tuberculosis infection and its diagnosis value

The aim was to establish a method of detecting tuberculosis infection by enzyme-linked immunospot （ELISPOT） assay and evaluate the application value of CFP10/ESAT6 fusion protein for diagnosis of tuberculosis （TB）. By comparing with the different experimental environment, the optimal detecting condition were identified including cell concentration, incubation period and protein antigen concentration, and ELISPOT assay was established. PPD skin test was set as control. It was found that the most appropriate conditions of ELISPOT assay were, 2 × 10^5 cell/each well, 20-40μg/mL CFP10/ESAT6 fusion protein as stimulus and 20 hours of incubation period. The positive rates of PPD skin test in 30 healthy persons, 38 patients with non-TB diseases and 40 TB patients were 40%, 0% and 72.5%, respectively. Whereas the positive rates of ELISPOT assay were 20.0%, 10.5% and 92.5 %, respectively. For the 40 TB patients, the sensitivities of ELISPOT in sputumpositive TB group and sputum-negative TB group were 94.4 % and 90.90%, respectively. It＇s concluded that ELISPOT could be used to detect tuberculosis infection efficiently and it might be an important assistant method in diagnosis of tuberculosis.