磁珠-流式细胞术检测肺结核患者Th1/Th2细胞因子

Cytokine bead assay for Th1 and Th2 cytokines in patients with pulmonary tuberculosis

目的建立磁珠-流式细胞术检测肺结核病患者血浆中的Th1/Th2细胞因子,为结核病患者免疫监测提供一种快速和客观的方法。方法按Th1/Th2细胞因子磁珠分析试剂盒说明书进行,Th1/Th2细胞因子标准品倍比稀释,50μL等体积混合的Th1/Th2细胞因子捕获磁珠加入检测管,依次加入50μL PE标记的检测试剂,50μL标准品或50μL待检样品震荡混匀,室温避光3 h,加1 mL洗液,200 g离心5 min,加300μL洗液悬浮磁珠,上流式细胞仪前各样品振荡3～5 s。采集流式图谱并用BD CBA software(version 4.0;BDBiosciences)软件分析,并换算成细胞因子浓度。结果涂阳肺结核患者和涂阴肺结核患者血浆中IFN-γ显著升高,而IL-5显著降低;涂阳肺结核患者循环IL-10显著升高,涂阴肺结核患者循环IL-10含量绝对值升高,但差异无显著性。结论与常规ELISA法相比,磁珠-流式细胞术检测细胞因子需更少的时间和样品。

【Objective】 To establish a speed and objective cytokine bead assay（CBA） to detect circulation Th1/Th2 cytokines in patients with pulmonary tuberculosis.【Methods】 The tests were completed according to Human Th1/Th2 Cytokine Kit Instruction Manual.50 μL the mixed capture beads were added to the assay tube,50 μL human Th1/Th2 PE detection reagent and 50 μL standard dilution or tested samples were sequentially added.The assay tubes were incubated for 3 hours at RT and protected from direct exposure to light.1 mL wash buffer was added to each assay tube,the tube was centrifuged at 200 g for 5 minutes and the supernatant was carefully discarded.The 300 μL wash buffer was added to each assay tube to resuspend the bead pellet.Before analyzing on the flow cytometer,each sample was vortexed for 3-5 seconds.The levels of cytokines were calculated using BD CBA software（version 4.0;BD Biosciences）.【Results】 The expression of IFN-γ and IL-10 in smear-positive pulmonary tuberculosis was significantly higher than that in control subjects,and the opposite was true for IL-5.IFN-γ expression was significantly higher in smear-negative pulmonary tuberculosis than in control subjects,while IL-5 followed the opposite directions.IL-10 expression was higher in smear-negative pulmonary tuberculosis than in control subjects,but there was no significant difference.【Conclusions】 Cytokine bead assay takes less time and less samples than conventional ELISA assays.