时间分辨免疫荧光法检测结核感染T细胞释放γ-干扰素的方法建立及初步临床应用

Establishment and Preliminary Clinical Application of Interferon Gamma Release Assays for T Cells Infected With Mycobacterium Tuberculosis Based on Time-Resolved Fluoroimmunoassay

目的建立一种检测结核感染T细胞释放γ-干扰素(IFN-γ)的时间分辨免疫荧光法(TRFIA)及初步临床应用。方法选择73例结核分枝杆菌阳性的肺结核患者,77例结核分枝杆菌阴性的肺结核患者,25例肺外结核患者,52例呼吸道疾病以外的非结核患者,70例非结核呼吸道疾病(含肺癌)患者作为研究对象。利用早期分泌抗原靶蛋白6(ESAT6)和培养滤液蛋白10(CFP-10)作为结核分枝杆菌特异性抗原,刺激患者全血标本中的结核分枝杆菌抗原特异性T细胞释放IFN-γ。植物血凝素L作为有丝分裂原,可非特异地刺激IFN-γ的产生。应用双抗体夹心TRFIA定量测定血浆中的IFN-γ浓度。评价TRFIA检测IFN-γ的精密度、检测低限、生物检测限、功能灵敏度、线性、准确度和特异度等分析性能指标,并与酶联免疫法(ELISA)进行比对试验研究。方法间结果差异比较采用χ~2检验,P<0.05为差异具有统计学意义。结果TRFIA检测IFN-γ的捕获抗体包被浓度为5.0μg/ml,生物素标记抗体使用工作稀释度为1∶1 800和铕标记物使用工作稀释度为1∶500;实验内变异系数(CV)<5%,实验间CV<10%;检测低限0.69pg/ml,生物检测限0.90pg/ml,功能灵敏度1.80pg/ml;线性范围2～5 000pg/ml;检测国际生物标准参考品的偏倚不超过5%;其他常见的细胞因子和常见内源性干扰物(胆红素、血红蛋白和三酰甘油)样本对该方法检测结果无明显影响。TRFIA检测297例临床样本结果判断与临床诊断的一致率可达85.19%;TRFIA与ELISA的结果具有高度一致性(κ=0.9 931),且TRFIA与ELISA的阳性检出率结果差异无统计学意义(χ~2=0,P>0.05)。结论 TRFIA检测IFN-γ的精密度好,灵敏度高,线性范围宽,准确度好,特异度强,临床符合率高,能满足临床检测需要。

Objective To establish an interferon gamma （IFN 7） release assays for T cells infected with mycobacterium tu berculosis based on time resolved fluoroimmunoassay（TRFIA） ,and evaluate its preliminary clinical application. Methods 73 patients with sputum smear-positive pulmonary tuberculosis, 77 patients with sputum smear negative pulmonary tuberculo sis, 25 patients with extrapulmonary tuberculosis, 52 patients with non tuberculosis and nowrespiratory diseases,and 70 pa dents with respiratory diseases （including lung cancer） and non tuberculous were selected as the study subjects. Early secre ring antigen target protein 6（ESAT6） and culture filter protein 10 （CFP 10） were used as mycobacterium specific antigen, which stimulate effector T cells of Mycobacterium tuberculosis antigen in patients peripheral blood. Phytohaemagglutinin L was mitogen and could stimulate the production of IFN -γ without selection. The concentration of IFN-γ was quantitatively determined by double antibody sandwich TRFIA. The precision,low limit of detection, biologic limit of detection, functional sensitivity, linear range, accuracy, specificity, and other analytical performance indicators of TRFIA were evaluated, and RFIA was compared with enzyme linked immunosorbent assay （ELISA）. The x2 test was used to test the difference between self-established method and the reference method of ELISA. If P value of X2 test was less than 0.05 ,the difference was sta tistically significant. Results The TRFIA capture IFN -γ antibody coating concentration was 5.0 μg/ml, the biotiwlabeled IFN -γ antibody working dilution was 1 ： 1 800 ,and the TRFIA europium marker working dilution was 1 ： 500. The intra as say and inter-assay coefficients of variation （CV） were less than 5% and 10% respectively, the low limit of detection was 0.69 pg/ml, the biologic limit of detection was 0.90 pg/ml, the functional sensitivity was 1.80 pg/ml, the linear range was 2 -5 000 pg/ml, the bias compared with international biological reference standard was less than 5 % and other cytokines and common endogenous interference （e. g. , bilirubin, hemoglobin and triglyceride） in the plasma did not interfere the detection results. The coincidence rate between the results of TRFIA and clinical diagnosis was 85.19%. The results of TRFIA and ELISA were highly consistent （k= 0.9 931）, and the positive detection rate was not statistically significant （x2 = 0, P〉0. 05）. Conclusion The TRFIA methodology has good precision, high sensitivity, wide linear range, good accuracy, strong specificity and high clinical compliance rate, which was valuable for clinical application.