A highly sensitive enzyme-linked immunosorbent assay for copper(II) determination in drinking water

Monoclonal antibodies against Cu(II)–EDTA were produced by using an immunogen of Cu(II) chelate conjugated to keyhole limpet hemocyanin (KLH) via a bifunctional chelator, 1-(4-isothiocyanobenzyl)ethylenediamine-N,N,N′,N′-tetraacetic acid (ITCBE). Stable hybridoma cell lines were generated with immunisation in mice followed by cell fusion. A monoclonal antibody with high affinity and specificity for Cu(II)-EDTA but not metal-free EDTA was produced from a selected hybridoma cell line (B11) and used to develop a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) for detection of Cu(II). The assay conditions, including the antigen coating, plate blocking and buffer pH, and ionic strength were examined for optimal assay performance. The optimised assay showed less than 3.9% cross-reactivities for Zn(II) and Co(II) and no cross-reactivities for other tested metal ions. The assay showed high sensitivity for Cu(II) with an IC₅₀ of 3.9 ng/mL, a detection limit of 0.24 ng/mL and a detection range of 0.67–29 ng/mL. Recoveries from drinking water were determined to be in the range of 94.4–117.2%.