Preparation and characterization of monoclonal antibody specific for copper-chelate complex

Monoclonal antibody (MAb) against chelated Cu(2+) was developed. The conjugate employed as immunogens in BALB/c mice to raise antibodies was synthesized using Cu(2+) coupling to keyhole limpet hemocyanin (KLH) via a bifunctional chelator (2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, p-SCN-Bn-DOTA). One of the hybridomas secreting antibody that bound tightly to Cu(2+)-ethylenediamine tetraacetic acid (EDTA) complex but not to metal-free EDTA was isolated from the fusion between murine splenocytes and SP2/0 myeloma cells. The performance of a competitive enzyme-linked immunosorbent assay (ELISA) was assessed for its sensitivity to changes in pH and ionic strength. The cross-reactivities (CR) with other metals were below 1%, except for Hg(2+) with a CR of 7.19 %. The IC(50) value achieved for copper was 0.89 μg/mL, showing a detection range of 0.25-8.13 μg/mL and the lowest detection limit of 0.032 μg/mL. The concentration of Cu(2+) in environmental water and serum samples obtained by ELISA correlated well with Atomic Absorption Spectrometry (AAS), and the mean recovery was 93.7%. These results indicated that ELISA could be a convenient analytical tool for monitoring copper in drinking water and human serum.