| 摘要: |
| 目的 基于γ干扰素释放试验（IGRA），利用流式荧光免疫分析法建立一种检测结核分枝杆菌（TB）感染的新方法。方法 分别用植物凝集素（PHA）、TB特异性混合多肽[早期分泌靶向抗原（ESAT-6）和培养滤液蛋白10（CFP-10）]刺激全血标本产生γ干扰素（IFN-γ），用双抗体夹心法结合流式荧光技术检测培养后血浆中的IFN-γ浓度，使用受试者工作特征（ROC）曲线评价其对TB感染的诊断效能。对该方法的线性范围、最低检测限、重复性、抗干扰性能及其与市售同类产品检测的一致性进行评价。结果 流式荧光免疫分析法检测IFN-γ的线性范围为2~1 000 pg/mL，最低检测限为0.3 pg/mL；检测100 pg/mL和500 pg/mL 2个浓度标本的重复性，2个浓度下变异系数分别为4.58%和2.46%；回收试验平均回收率为98.0%；三酰甘油≤ 50 mg/mL、胆红素≤ 0.6 mg/mL、血红蛋白≤ 10 mg/mL时对流式荧光免疫分析法检测结果无干扰。通过ROC曲线确定该方法诊断TB感染的最佳截断值为10 pg/mL，此时灵敏度为82.46%，特异度为87.30%。该方法与市售TB感染T淋巴细胞斑点试验（T-SPOT）试剂盒、QuantiFERON-TB Gold（QFT）试剂盒和万泰TB-IGRA试剂盒诊断TB感染的总符合率分别为92.5%、83.0%和85.4%，Kappa系数分别为0.822、0.622和0.630。结论 本研究建立的TB感染检测方法性能良好，准确性达到市售同类产品的水平，且在重复性、检测流程等方面具有明显优势。 |
| 关键词: 结核分枝杆菌 γ干扰素 流式荧光免疫试验 诊断 |
| DOI：10.16781/j.0258-879x.2019.07.0721 |
| 投稿时间：2019-02-18修订日期：2019-06-26 |
| 基金项目:上海青年临床医技人才（临床检验专业）培养资助计划（沪医卫基[2016]04号）. |
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| Flow cytometry fluorescence immunoassay based on interferon γ release assay: a method for Mycobacterium tuberculosis detection |
| ZHU Jun△,MA Wei△,LI Ya-zhou,LIU Yun,QIN Qin* |
(Department of Laboratory Medicine, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China
△Co-first authors.
* Corresponding author
*Corresponding author) |
| Abstract: |
| Objective To establish a new method for detecting Mycobacterium tuberculosis (TB) infection based on flow cytometry fluorescence immunoassay and interferon γ release assay (IGRA). Methods The whole blood samples were stimulated to produce interferon γ (IFN-γ) with phytagglutinin (PHA) and TB specific mixed peptides (early secretory antigenic target[ESAT-6] and culture filtrate protein 10[CFP-10]), and the plasma was analyzed by double antibody sandwich method combined with flow cytometry fluorescence immunoassay. The IFN-γ concentration was evaluated by receiver operating characteristic (ROC) curve for the diagnostic efficacy of TB. The linear range, minimum detection limit, repeatability, anti-interference performance of the established method were observed, and the consistency of detection with similar products on the market was evaluated. Results The linearity of the flow cytometry fluorescence immunoassay ranged from 2 pg/mL to 1 000 pg/mL. The lowest detection limit was 0.3 pg/mL; the repeatability parameters (coefficient of variation) of the samples at 100 pg/mL and 500 pg/mL were 4.58% and 2.46%, respectively. The average recovery rate of recovery assay was 98.0%. There was no interference with flow cytometry fluorescence immunoassay when the highest concentrations of triglyceride, bilirubin and hemoglobin were 50 mg/mL, 0.6 mg/mL and 10 mg/mL, respectively. As the optimum cut-off value of the IFN-γ concontration was 10 pg/mL, the sensitivity of IFN-γ in diagnosis of TB infection was 82.46% and the specificity was 87.30%. The total coincidence rates with T-SPOT, QFT, and Wantai TB-IGRA reagent were 97.2%, 83.0%, and 85.4%, respectively; and the Kappa coefficients were 0.822, 0.622 and 0.630, respectively. Conclusion The method for diagnosis of TB infection established in this study has a good performance, with the accuracy reaching the level of similar products on the market, and our method has obvious advantages in terms of repeatability and detection process. |
| Key words: Mycobacterium tuberculosis interferon γ flow cytometry fluorescence immunoassay diagnosis |
