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
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摘要:
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 9 ng/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& #177;8.65)ng/μmol Cr较运动前的(10.58& #177;5.62)ng/μmol Cr明显增加(P < 0.05); 吸烟群体 UMT含量为(86.58& #177;30.96)ng/μmol Cr, 显著高于不吸烟人群(14.73& #177;6.57)ng/μmol Cr(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。 Abstract: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 monoclonal antibody(McAb). 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& #177;5.62)ng/μmol Cr, while their UMT concentrations increased significantly after exercising, reached to(19.73& #177;8.65)ng/μmol Cr. UMT levels in smokers(86.58& #177;30.96)ng/μmol Cr were significantly higher than those in non-smokers(14.73& #177;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.
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