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 KinExATM 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.
Keywords: ELISA; KinExA; copper ions; drinking water; immunoassays.