流式微球分析技术联合检测血清MMP-9、MPO CD40L和t-PA的方法学建立及评价

Development and evaluation of a cytometric bead assay multiplex detection method for the detection of MMP-9,MPO,CD40L and t-PA

目的建立流式微球分析技术联合检测人血清中基质金属蛋白酶-9(MMP-9)、髓过氧化物酶(MPO)、CD40配体(CD40L)、血浆纤溶酶原激活物(t-PA)的方法并对其进行系统性评价。方法分别将四种选择好一定浓度的捕获抗体(MMP-9、MPO、CD40L和t-PA鼠抗人单克隆抗体)包被在四种已激活的不同荧光强度编码的羧基化聚苯乙烯微球上,用棋盘法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。结果反应体系中四种鼠抗人单克隆抗体最佳加入量为10μg,最佳生物素标记抗体浓度为1:4000倍稀释,最佳反应时间为2h,亲和素最适孵育时间为1h。MMP-9线性范围是0.20～333.33ng/mL,批内变异系数为4.60%～8.80%,批间变异系数为8.80%～9.60%,准确度相对偏倚为2.80%～3.18%,回收率是94.8～102.50%,灵敏度为0.20ng/mL;MPO线性范围是0.26～111.11ng/mL,批内变异系数为5.90%～7.70%,批间变异系数为9.10%～11.40%,准确度相对偏倚为1.44%～4.12%,回收率是97.8～103.2%,灵敏度为0.26ng/mL;CD40L线性范围是0.32～111.11g/mL,批内变异系数为5.40%～6.50%,批间变异系数为8.90%～12.40%,准确度相对偏倚为2.72%～5.95%,回收率是97.20～105.30%,灵敏度为0.32ng/mL;t-PA线性范围是1.21～111.11ng/mL,批内变异系数为2.20%～2.90%,批间变异系数为6.30%～12.20%,准确度相对偏倚为1.23%～4.60%,回收率是97.20～101.30%,灵敏度为1.21ng/mL。高浓度的甘油三酯、胆固醇和胆红素对四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MMP-9、MPO、CD40L和t-PA1流式微球联合检测技术,可拓展流式细胞分析技术,值得临床推广使用。

Objective To set up the cytometric bead assay multiplex detection and associate with the detection of MMP-9, MPO, CD40L and t-PA methodology and to evaluate the systematically Methods Four kinds of activated beads were respectively coated with a certain concentration of four kinds of capture antibody （mouse anti-human MMP-9 monoclonal antibody, mouse anti-human MPOmonoclonal antibody, mouse anti-human CD40L monoclonal antibody, mouse anti-human t-PA mono- clonal antibody）, and according checkerboard method to select the best test conditions, and applying the relevant rules of CLSI to methodology evaluation. Results In the reaction, the best quantity of the four kinds of mouse anti-human monoclonal antibody was 10tlg, the best dilution of biotin-labeled monoclonal antibody was 1：4000, the best incubation time was 2 hours, the best incubation time of streptavidin-PE was 1 hours. The linear range of MMP-9 was 0. 20-333. 33 ng/mL, the intra-assay of variation were 4.60O//oo and 8.80%, the inter-assay of variation were 8.80% and 9.60%, the relative bias were 2. 800//00 and 5.18%, the recovery were 94.8-102.5%, the sensitivity was 0.2 ng/mL. The linear range of MPO was 0. 26-111.11 ng/mL, the intra-assay of variation were 5.90% and 7.70%, the inter-assay of variation were 9.10% and 11.40%, the relative bias were 1.44% and 4.12%, the recovery was 97.8-103.2%, the sensitivity was 0.26 ng/mL. The linear range of CD40L was 0.32- 111.11 ng/mL, the intra-assay of variation were 5.40% and6.50 %, the inter-assay of variation were 8.90% and 12.40%, the relative bias were 2.72% and 5.95%, the recovery were 97.2-105.3%, the sensitivity was 0.32 ng/mL. The linear range of t-PA was 1.21-111.11 ng / mL, the intra-assay of variation were 2.20% and 2.90%, the inter-assay of variation were 6.30 % and 12.20%, the relative bias were 1.23% and 4.6%, the recovery was 97.2-101.3%, the sensitivity was 1.21 ng/mL, The high levels of triglyceride, cholesterol and bilirubin had certain interference to detection, the low levels of triglyceride, cholesterol and bilirubin had a minor disturbance, and there were no statistics signifi- cant differences with ELISA. Conclusion Fluorescent-bead-based multiplex detection method to detect MMP-9, MPO, CD40L and t-PA can be used in the clinical.