竞争性ELISA法检测人尿金属硫蛋白方法的改进及应用

Improvement and Application of a Competitive ELISA for Detection of Metallothionein in Human Urine

[目的]改进检测人尿金属硫蛋白(UMT)的竞争性酶联免疫吸附法(ELISA),探讨应用该法测定人尿样标本UMT含量的价值。[方法]分离纯化人肝脏金属硫蛋白(HMT),制备抗金属硫蛋白单克隆抗体(McAb),利用待测样品中MT与包被MT竞争结合McAb的原理,建立竞争性ELISA,并检测健康在校大学生298份尿样中的UMT含量。[结果]竞争性ELISA对人UMT最低检出限为9ng/mL,组内和组间的变异系数分别为1.78%～6.72%和5.45%～13.26%,样品的回收率为99.69%～107.25%。尿样检测结果显示运动后UMT含量为(19.73±8.65)ng/μmolCr较运动前的(10.58±5.62)ng/μmolCr明显增加(P<0.05);吸烟群体UMT含量为(86.58±30.96)ng/μmolCr,显著高于不吸烟人群[(14.73±6.57)ng/μmolCr](P<0.05)。[结论]本法对于检测人UMT具有较好的精密度和准确度,可用于人UMT检测;运动后UMT水平显著升高,吸烟者UMT明显高于正常对照。

[ Objective ] To improve and apply the competitive ELISA method for measuring human urinary metallothionein （ UMT ）. [ Methods ] Human metallothionein （ MT ） was isolated from an adult liver by SephadexG-75 and Sephadex G-50 chromatographic column, and then used to immunize Balb/c mice for preparing monoelonal antibody （ MeAb ）. The competitive ELISA was established with the McAb as the primary antibody. UMT was determined in 298 urine samples from university students. [Results] The detection limit of the ELISA was 9 ng/mL. The coefficients of variation for the within-running and between-running were 1.78%-6.72% and 5.45%-13.26%, respectively, while the recovery was 99.69%-107.25%. Before physical exercise, UMT concentrations of subjects were（ 10.58 ± 5.62 ）ng/μmol Cr, while their UMT concentrations increased significantly after exercising, reached to （ 19.73 ± 8.65 ） ng/μmol Cr. UMT levels in smokers （ 86.58 ± 30.96, ） ng/μmol Cr were significantly higher than those in non-smokers （ 14.73 ±6.57 ） ng/μmol Cr. [ Conclusion ] The competitive ELISA established was accurate and precise for measuring human UMT. The physical exercising and smoking influenced the UMT levels.