尿调蛋白酶联免疫吸附试验检测方法的建立及其在IgA肾病中的应用

摘要

目的:建立尿尿调蛋白的酶联免疫吸附试验(enzyme-linked immunosorbent assays,ELISA)检测方法,并对IgA肾病患者的尿尿调蛋白水平监测进行进一步验证.方法:以多克隆抗体作为包被抗体,单克隆抗体作为检测抗体,建立尿调蛋白的快速双抗夹心检测方法,检测其精确性及重复性,随机选取55例尿液标本,同时以商品化试剂盒与本实验方法进行检测,比较166例IgA肾病患者和正常人的尿尿调蛋白水平.结果:获得的标准曲线为0.78 ～ 12.5 μg/L,实验室内变异系数为7.5％,实验室间变异系数为7.9％.55例尿液标本的商品化试剂盒与本实验方法结果比对,相关系数为r=0.615,P＜O.001；166例IgA肾病患者的尿尿调蛋白/尿肌酐比值低于正常人.结论:尿尿调蛋白的ELISA检测方法灵敏,重复性较好,可运用于大样本的人群检测,IgA肾病患者的尿尿调蛋白分泌低于正常人.%Objective: To establish a method of enzyme-linked immunosorbent assay ( El,ISA) to measure urinary uromodulin and explore the urinary uromudulin level in IgA nephropathy. Methods; The rabbit anti-human uromodulin polyclonal antibodies were coated on plates to capture uromodulin and the mouse anti-human uromodulin monoclonal antibody was used as detecting antibody to set up ELISA procedure. The precision and repeatability of this ELISA method were evaluated, and then this method was compared with the commercialized Tamm-Horsfall Glycoprotein ELISA Kit by examining urinary uromodulin levels in 55 individuals. Finally, the urinary uromodulin level in 166 IgA nephropathy patients were detected as well as 48 normal controls with this established method. Results; The detecting range of uromodulin was 0. 78 - 12. 5 |Ag/L by this method. The coefficient of variation within-run was 7. 5% , and between-run of coefficient of variation was 7. 9%. Correlation of this method and comercialized kit was good (r =0.615, P <0. 001). The urinary uromodulin/urinary creatinine ratio in IgA nephropathy was significantly lower than that in normal controls. Conclusion; The established ELISA method is sensitive and repeatable, and can be used in further studies.