环境水样中六价铬酶联免疫检测方法的建立.docVIP

 环境水样中六价铬酶联免疫检测方法的建立# 邹军辉，唐勇，钟辉，宋建勋，欧子强** （暨南大学 广东省分子免疫和抗体工程重点实验室，广州 510632） 5 10 15 20 25 30 35 40 摘要：以抗 Cr3+-EDTA 单克隆抗体为基础，建立优化的间接竞争 ELISA 方法和样品前处理 方法，用于环境水样中 Cr6+的检测。分别配制不同 pH 及不同离子强度的 PBS 保温液，优化 筛选 ELSIA 的最佳反应 pH 值与离子强度。前处理过程中，通过调节样品 pH，沉淀去除水 样原有 Cr3+；优化各项还原条件使 Cr6+还原为 Cr3+，调节 pH 进行 ELISA 检测，通过检测出 的 Cr3+的量计算原有 Cr6+的含量。结果表明，当 ELISA 保温液 pH 为 7.4，离子强度为 0.15M 时，ELISA 的灵敏度（IC50）最佳，为 5.9 ng/mL，检测限为 0.35 ng/mL，线性范围是 0.35~50 ng/mL。当还原剂为 60 μg/mL NaHSO3，还原 pH 为 3，反应时间为 15 min 时样品前处理效 率最高。以结合最优样品前处理方法的 ELISA 检测实际水样并与 ICP 检测结果比较，得到 两种方法检测结果数据的相关系数达 0.9887，表明本研究建立的 ELISA 方法检测结果可靠。 加标实验结果显示该 ELSIA 方法变异系数小于 12%，说明该方法具有良好的重复性和精密 度，可用于环境水样六价铬离子的监测。 关键词：六价铬；ELISA；环境水样 中图分类号：R392.11 Development of enzyme-linked immunoassay for hexavalent chromium detection in environmental water samples ZOU Junhui, Tang Yong, ZHONG Hui, SONG Jianxun, OU Ziqiang (Guangdong Province Key Laboratory of Molecular Immunology and Antibody Engineering, Jinan University, Guangzhou 510632) Abstract: An optimal indirect competitive ELISA was developed using monoclonal antibody against Cr3+-EDTA. The pretreatment procedure was also established. Reaction conditions, such as pH and ionic strength, were studied to optimize the immunoassay. In pretreatment procedure, the Cr3+ existent in samples was precipitated by adjusting pH and the reducing conditions of Cr6+ were researched completely, the concentration of Cr6+ in samples were calculated as Cr3+ detected by ELISA. Results showed that the ELISA had the most sensitive to soluble Cr3+-EDTA when pH was 7.4 and ionic strength was 0.15 M, the IC50 and limit of detection was 5.9ng/mL, 0.35ng/mL, respectively, and the liner range was 0.35~50ng/mL. The collected supernatant containing Cr6+ was mixed with 60mg/L of NaHSO3 solution, the Cr6+ was reduced to Cr3+ at pH 3.0 and reacted for 15 min. Finally, ELISA was used to detect the concentration of Cr6+ in samples. The correlation coefficient between the ELISA and ICP was 0.9887 and the coefficient of variation