血栓调节蛋白化学发光免疫分析检测方法的建立*

目的:建立并评价基于板式化学发光免疫分析(CLIA)平台的血栓调节蛋白(TM)定量检测方法。方法:以链霉亲和素包被微孔板,加入待检血浆,偶联生物素和辣根过氧化物酶的配对抗体组成分析体系,采用双抗体夹心模式,建立TM抗原定量检测方法,并对其进行条件优化和性能评价。结果:生物素化抗体和酶标抗体的工作浓度分别为0.5 μg/mL和0.75 μg/mL,加样后的孵育时间选为15 min,最低检测限为0.2 TU/mL,该检测方法的检测范围为1~200 TU/mL,批间和批内精密度(CV)均小于8%,37 ℃ 10天稳定性良好,207份临床血浆测值与希森美康测值相关性较高(R²>0.96)。结论:建立了TM板式化学发光定量检测方法,且各项性能指标良好,可满足临床检测的需要。

The Development of Chemiluminescence Immunoassay Detection Method for Thrombomodulin

Objective: To establish and evaluate a quantitative detection method for thrombomodulin (TM) based on a plate chemiluminescence immunoassay (CLIA) platform. Methods: The analysis system was composed of microplate coated with streptavidin, serum, the paired antibody coupled biotin and horseradish peroxidase. The double antibody sandwich mode was adopted to test quantitatively TM antigen. Moreover,the reaction conditions of TM were optimized and the analytical performance was evaluated. Results: The working concentration of biotinylated antibody and enzyme-labeled antibody was 0.5 μg/mL and 0.75 μg/mL, respectively. The incubation time was 15 min; the minimum detection limit was 0.2 TU/mL and the range of the detection method was 1~200 TU/mL. Inter-assay and intra-assay precision (CV) were less than 8%. The established method had good stability during 10 days at 37 ℃. 207 clinical plasma measured values had a high correlation with Sysmex measured values (R²>0.96). Conclusion: This study has successfully established a quantitative detection method of TM plate chemiluminescence, which has good performance indicators and can meet the needs of clinical detection.