Development Of Immunoassay For The Determination Of Cadmium

An indirect competitive enzyme-linked immunosorbent assay (icELISA) and a colloidal gold-based immunochromatographic strip were systematacially developed to detect the heavy metal cadmium residues.In the assay, we use the bifunctional chelating agent1-(4-isothiocyanobenzyl) ethylenediamine-N,N,N’,N-tetraacetic acid (ITCBE) to chelate cadmium and conjugatethe protein (KLH and BSA), and make successfully the immunogen and coating antigen. New Zealand white rabbits were immunized and the polyclonal antibody was obtained from its serum with high specific against cadmium.Under the optimized coating antigen concentration, antibody dilution times, ionic strength, pH value and blocking buffer, a standard inhibition curve for cadmium in PBS was performed. The ELIS A had a half-maximum inhibition concentration (IC50) of4.18μ.g/L and a limit of detection (LOD, calculated as IC15value) of0.38μg/L. The results showed that the cross-reactivities of the all metal chelates were less than0.3%, except for the mercury-chelate (the cross-reactivity rate was10.6%) and chromium-chelate (the cross-reactivity rate was1.8%). Traditional Chinese medicine A. dahurica (Fisch.) Benth. et Hook, tap water and river water samples were selected as the object of the study to detect the recoveries of the spiked cadmium in samples. The results demonstrated that the average recoveries rate in samples were90.54%-115.36%, and the coefficients of variation were1.35%-9.33%. The spiked samples were simultaneously analyzed by inductively coupled plasma mass spectrometry (ICP-MS) to further verify the accuracy of ELISA. The regression equation in traditional Chinese medicine A. dahurica (Fisch.) Benth. et Hook sample was y=1.087x-0.029, and the correlation coefficients were0.994. The regression equation in water samples was y=0.987x-0.085, and the correlation coefficients were0.986. The obtained results indicated that the developed ELISA had a favorable accurate and reliable and it is suitable for the analysis of heavy metal cadmium residues in traditional Chinese medicine and water samples.Meanwhile, an immunochromatographic strip assay using colloidal gold-labeled antibody was developed. A mean diameter of20nm colloidal gold particles was prepared using sodium citrate method. Under optimal conditions, the colloidal gold-antibody conjugate was prepared and sprayed onto a conjugate pad. The coating antigen as test line (T line) and goat anti-rabbit IgG as control line (C line) were separately immobilized on nitrocellulose (NC) membrane. The visual LOD (the minimum concentration producing the color on the test line significantly weaker than that on the negative control) was5μg/L. Tap water and river water samples were analyzed directly by the test strip without any pretreatment and dilution procedure. Then the results were compared to the ELISA analysis. The results obtained by the immunochromatographic strip correlated well with those determine by ELISA. The method had high sensitivity, rapid detection, without any pretreatment process, and it is suitable for on-site monitoring of the cadmium in water samples.