流式微球检测超敏C反应蛋白方法学的建立及性能评价

目的建立流式微球分析技术（CBA）检测人血清中超敏C反应蛋白（hs—CRP）的方法并对其进行系统性评价。方法将hs—CRP鼠抗人单克隆抗体包被在已激活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，并应用美国临床实验室标准化协会的有关规则进行方法学评价。结果试验选择hs—CRP鼠抗人单克隆抗体的加入量为10μg，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2次，PE标记亲和素的稀释倍数为1：1000，第2次反应1h洗涤1次。方法学评价显示，CBA检测hs—CRP的分析灵敏度为0．03ng／mL，线性范围为0．03～333．33ng／mL，批内变异系数为2．70％～4．44％，批间变异系数为4．99％～9．52％，准确度相对偏倚为2．17％～4．39％，回收率为98．31％～104．60％。高浓度的三酰甘油、胆固醇和胆红素对3种因子有一定的干扰率，低浓度的三酰甘油、胆固醇和胆红素对3种因子干扰较小。分别与免疫荧光方法比较差异无统计学意义。结论自建的hs—CRP流式微球检测技术性能指标满足公认的质量指标，可拓展流式细胞分析技术，值得临床推广使用。

Development and evaluation of flow cytometric bead assay detection method for the detection of hs CRP

Objective To develop and systematically evaluate the flow cytometric bead analysis technique for the detection of high sensitivity C reactive protein. Methods The mouse anti human hs CRP monoclonal antibody was coated on the activated carboxylated polystyrene beads. According to the orthogonal experiment, the best test conditions was selectand and the relevant rules of CLSI was applied for methodology evaluation. Results In the reaction, the best quantity of mouse anti human hs CRP monoclonal antibody was 10μg,the best dilution of biotin labeled monoclonal antibody was 1 ： 540. The first incubation time was 2 hours with twice of washing. The best dilution of streptavidin PE was 1 ： 1 000, and the second incubation time of streptavidin PE was 1 hours with one times of washing. According the methodology evaluation, the sensitivity of hs-CRP was 0. 03 ng/mL, the linear range was from 0.03 to 333.33 ng/mL,the intra assay of variation was from 2.70% to 4.44% ,the inter assay of variation was from 4.99% to 9.52% ,the relative bias was from 2.17% to 4.39% ,and the recovery was from 98.31% to104.60%. High levels of triglyceride,cholesterol and bilirubin had a certain interference to detection, but low levels of triglyceride,cholesterol and bilirubin had a minor disturbance. There was no significant difference with immunofluorescenee. Conclusion The performance indicators of cytometric bead assay of detecting hs CRP meet the recognized quality indicators,and the method could extend flow eytometry analysis,worthy of clinical use.