褐牙鲆卵黄原蛋白免疫胶体金技术的开发及其在环境雌激素检测中的应用

Development of Lateral Flow Immunoassay for Paralichthys olivaceus Vitellogenin and Its Application in Detecting Environmental Estrogen

为快速检测海洋环境雌激素,本研究利用20 nm胶体金颗粒和褐牙鲆(Paralichthys olivaceus)Vtg单/多克隆抗体开发了夹心法免疫胶体金检测试纸条,并对胶体金标记条件、T线和C线抗体浓度等参数进行了优化,构建了褐牙鲆Vtg的快速检测技术。实验结果表明该胶体金试纸条工作范围为15.7~1000 ng·mL^(-1)(R^(2)>0.98),检出限为7.8 ng·mL^(-1)。本研究制备的胶体金试纸条检出限与目前常用的多种Vtg的ELISA方法相近,但是检测时间仅为15 min,比常规方法节省了约14 h。此外,该试纸条在存放30和90 d后,对Vtg的检测结果并无明显偏差,表现出较好的稳定性和可重复性。最后,通过测定多种常见环境雌激素(双酚A、雌二醇、乙炔雌二醇)暴露对雄性成年褐牙鲆血浆中Vtg的诱导情况,进一步验证了该试纸条在海洋环境雌激素活性检测中的应用。

In order to rapidly detect estrogens in the marine environment,a lateral flow immunoassay was developed based on 20 nm colloidal gold particles and monoclonal/polyclonal antibodies against Paralichthys olivaceus Vtg.The labeling conditions of colloidal gold and concentrations of T and C line antibodies were optimized to obtain the best detection performance.Under the optimal conditions,the working range of lateral flow immunoassay was 15.7~1000 ng·mL^(-1),and the limit of detection(LOD)was 7.8 ng·mL^(-1),which is similar to those of the previously reported enzyme linked immunosorbent assays(ELISA)for fish Vtgs.Meanwhile,an advantage of this method was its rapid processing time and the assay could be completed within 15 min.In addition,there was no significant difference in the detection results of lateral flow immunoassay after storage for 30 days and 90 days,indicating that the lateral flow immunoassay had good stability and repeatability.Finally,the usefulness was testified by quantifying Vtg concentrations in plasma of adult Paralichthys olivaceus exposed to bisphenol A,estradiol and ethinylestradiol,respectively.These results suggested that this study provided a rapidly and accurate detection method for marine environmental estrogen.