Monoclonal Antibody to Trivalent Chromium Chelate Complex and Its Application to Measurement of the Total Chromium Concentration

Isothiocyanobenzyl group-appended ethylenediamine tetraacetic acid (EDTA) was used to covalently couple Cr(III)center dot EDTA to keyhole limpet hemocyanin for use as an immunogen. An obtained monoclonal antibody (RD3G4) bound to Cr(III)center dot EDTA with an equilibrium dissociation constant (K(d)) of 9.7 nM, which was 100-fold tighter than the K(d)(S) for the other tested EDTA-metal complex. In particular, there was an over 2000-fold affinity difference between Cr(III)center dot EDTA and Fe(III)center dot EDTA, although the ion radius of trivalent chromium (0.76 A) was quite close to that of ferric ion (0.79 angstrom). Hexavalent chromium could be detected by the antibody after being reduced into trivalent form. An immunoassay format showed an IC(50) of 87 nM for hexavalent chromium, with a detection limit of 30 nM (1.6 mu g/L). Therefore, the addition of reducing agents to the mixture of tri- and hexavalent chromium allows determination of the total chromium concentration by the immunoassay. Hexavalent chromium could be isolated from trivalent chromium by an anion-exchange column, and thus, the concentration of hexavalent chromium in tri- and hexa- mixture can also be estimated by the immunoassay.