Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh P.O. Box 2457 11451, Saudi Arabia.
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Molecules. 2023 Oct 10;28(20):7017. doi: 10.3390/molecules28207017.
This study describes the development of two highly sensitive immunosensor platforms for the trace determination of copper ions, Cu(II), in drinking water. These platforms were a microwell-based enzyme-linked immunosorbent assay (ELISA) and a kinetic exclusion assay (KinExA) with a KinExA 3200 immunosensor. Both ELISA and KinExA were developed utilizing the same antibody and coating reagent. The antibody was a mouse monoclonal antibody, designated as 8D66, that specifically recognized Cu(II)-ethylenediamine tetraacetic acid complex (Cu(II)-EDTA) but did not recognize Cu(II)-free EDTA. The 8D66 monoclonal antibody was generated by the fusion of spleen cells of an immunized BALB/c mouse with SP2/0-Ag14 myeloma cells. The immunogen was a protein conjugate of Cu(II)-EDTA with keyhole limpet hemocyanin protein. The coating reagent was Cu(II)-EDTA covalently linked to bovine serum albumin protein (Cu(II)-EDTA-BSA). Both assays involved the competitive binding reaction between Cu(II)-EDTA complexes, formed in the sample solution, and Cu(II)-EDTA-BSA conjugate which has been immobilized onto ELISA plates (in ELISA) or polymethylmethacrylate beads (in KinExA) for a limited quantity of binding sites of the 8D66 antibody. In ELISA, color signals were generated by a peroxidase-labeled secondary antibody and 3,3',5,5'-tetramethylbenzidine substrate. In KinExA, a fluorescein isothiocyanate-labeled secondary antibody was used to generate KinExAgram (trend-line fluorescence responses vs. time). The conditions of both ELISA and KinExA were investigated, and the optimum procedures were established. Both ELISA and KinExA were validated, and all validation parameters were acceptable. Many different metal ions that are commonly encountered in drinking water did not interfere with the Cu(II) analysis by both ELISA and KinExA. Both assays were applied to the determination of Cu(II) in drinking water with satisfactory accuracy and precision. Both assays were compared favorably with inductively coupled plasma atomic emission spectroscopy in terms of their abilities to accurately and precisely determine Cu(II) in drinking water samples. A comparative evaluation of ELISA and KinExA revealed that KinExA had a higher sensitivity and better precision than ELISA, whereas both assays had comparable accuracy. Both ELISA and KinExA were superior to the existing atomic spectrometric methods for Cu(II) in terms of sensitivity, convenience, and analysis throughputs. The proposed ELISA and KinExA are anticipated to effectively contribute to assessing Cu(II) concentrations and control the exposure of humans to its potential toxicities.
本研究描述了两种用于痕量测定饮用水中铜离子（Cu(II)）的高灵敏度免疫传感器平台的开发。这两个平台分别是基于微孔的酶联免疫吸附测定（ELISA）和动力学排除测定（KinExA），以及 KinExA 3200 免疫传感器。ELISA 和 KinExA 均利用相同的抗体和包被试剂开发。该抗体是一种鼠单克隆抗体，命名为 8D66，它特异性识别 Cu(II)-乙二胺四乙酸络合物（Cu(II)-EDTA），但不识别 Cu(II)-游离 EDTA。8D66 单克隆抗体由免疫 BALB/c 小鼠的脾细胞与 SP2/0-Ag14 骨髓瘤细胞融合产生。免疫原是 Cu(II)-EDTA 与贻贝血红蛋白蛋白的蛋白缀合物。包被试剂是 Cu(II)-EDTA 与牛血清白蛋白蛋白（Cu(II)-EDTA-BSA）共价结合。两种测定均涉及样品溶液中形成的 Cu(II)-EDTA 络合物与固定在 ELISA 板上的 Cu(II)-EDTA-BSA 缀合物（在 ELISA 中）或聚甲基丙烯酸甲酯珠（在 KinExA 中）之间的竞争结合反应，用于 8D66 抗体的有限数量的结合位点。在 ELISA 中，通过辣根过氧化物酶标记的二抗和 3,3',5,5'-四甲基联苯胺底物产生颜色信号。在 KinExA 中，使用荧光素异硫氰酸酯标记的二抗生成 KinExAgram（趋势线荧光响应与时间）。对 ELISA 和 KinExA 的条件进行了研究，并确定了最佳程序。对 ELISA 和 KinExA 进行了验证，所有验证参数均合格。许多在饮用水中常见的不同金属离子不会干扰 ELISA 和 KinExA 对 Cu(II)的分析。两种测定均应用于饮用水中 Cu(II)的测定，结果令人满意，精密度和准确度均较高。与电感耦合等离子体原子发射光谱法相比，这两种测定方法在准确、精确地测定饮用水样品中的 Cu(II)方面表现出色。ELISA 和 KinExA 的比较评估表明，KinExA 比 ELISA 具有更高的灵敏度和更好的精密度，而两种测定方法的准确性相当。在灵敏度、便利性和分析通量方面，ELISA 和 KinExA 均优于现有的原子光谱法测定 Cu(II)。预计提出的 ELISA 和 KinExA 将有效用于评估 Cu(II)浓度，并控制人类接触其潜在毒性的风险。
