Cadmium purification and quantification using immunochromatography.

One of the pathways by which cadmium enters human beings is through the consumption of agricultural products. The monitoring of cadmium has a significant role in the management of cadmium intake. Cadmium purification and quantification using immunochromatography were conducted in this study as an alternative means of cadmium analysis. The samples used in this study were rice, tomato, lettuce, garden pea, Arabidopsis thaliana (a widely used model organism for studying plants), soil, and fertilizer. The cadmium immunochromatography has been produced from the monoclonal antibody Nx2C3, which recognize the chelate form of cadmium, Cd.EDTA. The immunochromatography can be used for quantification of cadmium in a range from 0.01 to 0.1 mg/L at 20% mean coefficient of variance. A chelate column employing quaternary ammonium salts was used for the purification of cadmium from HCl extracts of samples. Recoveries of cadmium were near 100%, and the lowest recovery was 76.6% from rice leaves. The estimated cadmium concentrations from the immunochromatography procedure were evaluated by comparison with the results of instrumental analysis (ICP-AES or ICP-MS). By comparison of HCl extracts analyzed by ICP-MS and column eluates analyzed by immunochromatography of the samples, the estimated cadmium concentrations were closely similar, and their recoveries were from 98 to 116%.

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) x EDTA to keyhole limpet hemocyanin for use as an immunogen. An obtained monoclonal antibody (RD3G4) bound to Cr(III) x 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) x EDTA and Fe(III) x EDTA, although the ion radius of trivalent chromium (0.76 A) was quite close to that of ferric ion (0.79 A). Hexavalent chromium could be detected by the antibody after being reduced into trivalent form. An immunoassay format showed an IC50 of 87 nM for hexavalent chromium, with a detection limit of 30 nM (1.6 microg/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.

Simple method to reduce interference from excess magnesium in cadmium immunoassays.

In order to develop a rapid inexpensive test for cadmium in rice, we identified an antibody specific for cadmium-EDTA complexes; this antibody binds to cadmium-EDTA with a Kd of approximately 10(-8) M. Although the antibody's cross reactivity to magnesium was minimal (Kd approximately 10(-5) M), the high toxicity of cadmium coupled with the high natural occurrence of magnesium in rice resulted in a situation where magnesium interfered with cadmium determination and resulted in falsely elevated estimates of cadmium. Fortunately, the formation constant of EDTA for cadmium is approximately 5 x 10(7) times higher (at pH 7) than the formation constant of EDTA for magnesium, and we were able to eliminate the magnesium interference by judicious selection of the EDTA concentration used in the assay. The resulting equilibria are complex, but we show that a relatively simple two-step model in which cadmium and magnesium compete for EDTA followed by cadmium-EDTA and magnesium-EDTA competing for antibody provided a good fit to the measured data. These analyses enabled appropriate selection of the optimum EDTA concentration for an immunoassay with improved selectivity.