流式微球联合检测炎症性因子

流式微球联合检测炎症性因子

IL-6、TNF-α和MCP-1方法学的建立和评价

令狐颖1张程2陈艳2 黄山1* 许健1刘志琴3

（1.贵州省人民医院临床检验中心，贵州贵阳 550002；2.贵阳医学院09级

硕士研究生；3.本院心内科）

【摘要】目的建立流式微球分析技术联合检测人血清中细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）和单核细胞趋化因子-1（MCP-1）的方法并对其进行系统性评价。方法分别将三种选择好一定浓度的捕获抗体（IL-6、TNF-α和MCP-1鼠抗人单克隆抗体）包被在三种已激活的不同荧光强度编码的羧基化聚苯乙烯微球上，用正交试验对试验条件进行优化选择，并应用CLSI的有关规则进行方法学评价。结果实验选择各种捕获单克隆抗体的加入量为10µg，各种生物素标记的羊抗人单克隆抗体的稀释倍数为1:540，第一次反应时间为2h、洗涤二次，PE标记亲和素的稀释倍数为1:1000，第二次反应1h洗涤一次。方法学评价显色：IL-6线性范围是1.13—3333.33 pg/mL，批内变异系数为1.98%—2.31%，批间变异系数为3.52%—4.17%，准确度相对偏倚为0.19%—0.66%，回收率是95.4—101.5%，灵敏度为1.13 pg/mL；TNF-α线性范围是1.07—1111.11 pg/mL，批内变异系数为2.67%—2.90%，批间变异系数为4.27%—4.91%，准确度相对偏倚为0.85%—1.53%，回收率是97.3—104.2%，灵敏度为1.07 pg/mL；MCP-1线性范围是7.07—3333.33 pg/mL，批内变异系数为2.63%—2.92%，批间变异系数为3.70%—4.23%，准确度相对偏倚为0.15%—1.06%，回收率是95.2—104.3%，灵敏度为7.07 pg/mL.高浓度的甘油三酯、胆固醇和胆红素对三种因子有一定的干扰率，低浓度的甘油三酯、胆固醇和胆红素对三种因子干扰较小。分别与ELISA方法比较无显著差异。结论自建的IL-6、TNF-α和MCP-1流式微球联合检测技术，可拓展流式细胞分析技术，值得临

基金项目：贵阳市科技局社会发展领域科技攻关项目[2010]筑科农合同字第1-社-35号和贵州省卫生厅立项资助项目[Gzwlj2009-1-007]。

*通讯作者。

床推广使用。

【关键词】流式微球分析技术（CBA）；联合检测；细胞介素-6（IL-6）；肿瘤坏死因子-α（TNF-α）；单核细胞趋化因子-1（MCP-1）)

Development and evaluation of a Cytometric Bead Assay Multiplex Detection Method for the detection of IL-6、TNF-αand MCP-1

LING-Hu ying1,HUANG Shan1，ZHANG Cheng2, CHEN Yan2, XU Jian1，LIU Zhi-qin3

(1.Guizhou Province Center for Clinical Laboratory;2.Guiyang Medical College;3.Guizhou Province People's Hospital)

【Abstract】Objective To development and Systematic review of a cytometric bead array Multiplex Detection Method for the detection of IL-6, TNF-α and MCP-1. Method A certain concentration of three kinds of capture antibody (mouse anti-human IL-6 monoclonal antibody, mouse anti-human TNF-α monoclonal antibody, mouse anti-human MCP-1 monoclonal antibody) were respectively coated with three kinds of activated beads, and according orthogonal experiment to select the best test conditions, and applying the relevant rules of CLSI to methodology evaluation. Result In the reaction, the best quantity of the three kinds of mouse anti-human monoclonal antibody was 10μg, the best dilution of biotin-labeled monoclonal antibody was 1:540, the best incubation time was 2 hours, the first washing time was 2, the beat dilution of streptavidin-PE was 1:1000, the best incubation time of streptavidin-PE was 1 hours, the second washing time was 1. According the methodology evaluation, the linear range of IL-6 was 1.13-3333.33 pg / ml, the intra-assay of variation was 1.98% and 2.31%, the inter-assay of variation

was 3.52% and 4.17%, the relative bias was 0.19% and 0.66%, the recovery was 95.4-101.5%, the sensitivity was 1.13 pg/ml; the linear range of TNF-α was 1.07-1111.11 pg / ml, the intra-assay of variation was 2.67% and 2.90%, the inter-assay of variation was 4.27% and 4.91%, the relative bias was 0.85% and 1.53%, the recovery was 97.3-104.2%, the sensitivity was 1.07 pg/ml; the linear range of MCP-1 was 7.07-3333.33 pg / ml, the intra-assay of variation was 2.63% and 2.92%, the inter-assay of variation was 3.70% and 4.23%, the relative bias was 0.15% and 1.06%, the recovery was 95.2-104.3%, the sensitivity was 7.07 pg/ml. The high levels of triglyceride, cholesterol and bilirubin had a certain interference to detection, the low levels of triglyceride, cholesterol and bilirubin had a minor disturbance, and it had no significant difference with ELISA. Conclusion Cytometric bead array multiplex detection method to detect IL-6, TNF-α and MCP-1 can be used in the clinical and expansion of flow cytometry.

【Key words】Cytometric bead array; Multiplex detection; IL-6、TNF-α;MCP-1.

目前，冠心病（coronary artery heart disease, CHD）已成为危害我国人民群众生命和健康的重大疾病[1]。近年来的研究，明确了血管壁的炎症性变化在冠状动脉粥样硬化及其并发症的发生进展过程中发挥了非常大的作用。许多炎症性因子，如白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）和单核细胞趋化因子-1（MCP-1）等炎性因子参与了动脉粥样硬化的形成过程。随着流式细胞技术的不断普及，对同一标本进行多参数的流式微球联合检测，具有广阔的应用前景。本文利用微球荧光编码技术，结合流式细胞分析技术，建立了IL-6、TNF-α和MCP-1的流式微球联合检测分析技术，具有灵敏度高，特异性强，节约成本等特点。

1 材料和方法

1.1 标本来源

血浆标本采自2010年9月—2011年3月贵州省人民医院心内科已确