Simplified Mercury Extraction from Coal Fly Ash for Quantification of Total Mercury by ELISA-based Immunoassay.

A simplified two-step mercury extraction procedure enabled the selective and reproducible mercury recovery from actual coal fly ash (CFA). The optimized extraction procedure involving conventional enzyme-linked immunosorbent assay (ELISA)-based immunoassay allowed the ultra-sensitive quantification of total mercury content in CFA. The total mercury content of 41 CFA samples were successfully determined using the above-mentioned method, and the results were in agreement with those obtained by standard instrumental analysis (thermal decomposition atomic absorption spectrometry) within a 15% coefficient of variation. Our method for total mercury quantification is not only simple but suitable for management of the mercury content at coal-fired electric power plants and landfill sites, which deal with large amounts of waste CFA.

Label-free impedimetric immunoassay for trace levels of polychlorinated biphenyls in insulating oil.

A rapid, ultrasensitive, and practical label-free impedimetric immunoassay for measuring trace levels of total polychlorinated biphenyls (PCBs) in insulating oil was developed. First, we developed a novel monoclonal antibody (RU6F9) for PCBs by using a designed immunogen and characterized its binding affinity for a commercial mixtures of PCBs and its main congeners. A micro comblike gold electrode was fabricated, and the antibody was covalently immobilized on the electrode through a self-assembled monolayer formed by dithiobis-N-succinimidyl propionate. The antigen-binding event on the surface of the functionalized electrode was determined as the change in charge transfer resistance by using electrochemical impedance spectroscopy. The resulting impedimetric immunoassay in aqueous solution achieved a wide determination range (0.01-10 µg/L) and a low detection limit (LOD) of 0.001 µg/L, which was 100-fold more sensitive than a conventional flow-based immunoassay for PCBs. By combining the impedimetric immunoassay with a cleanup procedure for insulating oil utilizing a multilayer cleanup column followed by DMSO partitioning, an LOD of 0.052 mg/kg-oil was achieved, which satisfied the Japanese regulation criterion of 0.5 mg/kg-oil. Finally, the immunoassay was employed to determine total PCB levels in actual used insulating oils (n = 33) sampled from a used transformer containing trace levels of PCBs, and the results agreed well with the Japanese official method (HRGC/HRMS).

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.