Preparation of Specific Monoclonal Antibodies (MAbs) against Heavy Metals: MAbs That Recognize Chelated Cadmium Ions

Monoclonal antibodies (MAbs) were produced against chelated Cd (2+). Since Cd (2+) ions are too small to elicit an immune response, the metal was coupled to protein carrier (keyhole limpet hemocyanin, KLH) using a bifunctional chelator 1-(4-isothiocyanobenzyl)ethylenediamine N, N, N', N'-tetraacetic acid (ITCBE). Several mice were immunized with this Cd (2+)-ITCBE-KLH immunoconjugate. Spleen cells of two immunized mice were fused with myeloma cells, and the resulting hybridomas were screened using protein conjugates with covalently bound metal-free ITCBE (ethylenediamine tetraacetic acid) or Cd (2+)-ITCBE. Four hybridoma cell lines that produced MAbs with high selectivity and sensitivity (Aa4, Aa6, Ac4, and Ba2) were expanded for further study. Cross-reactivities with other metals were below 1% except for Hg (2+), which showed a slight cross-reactivity in competitive ELISA. These antibodies were used to construct competitive ELISAs for ionic cadmium; the IC 50 of the four antibodies (Aa4, Aa6, Ac4, and Ba2) were 10.59, 4.19, 29.45, and 6.63 microg/L, respectively. The detection range and the lowest detection limit for cadmium, using the Aa6 antibody, were 2.19-86.38 microg/L and 0.313 microg/L, respectively. Spike-recovery studies in tap and stream water showed that the most sensitive antibody can be used for cadmium detection in drinking water.