Development and Validation of a One-Step Immunoassay for Determination of Cadmium in Human Serum

Abstract

A sensitive and simple one-step immunoassay was developed and validated for quantitative determination of Cd(II) in human serum. In this method, a monoclonal antibody that recognizes Cd(II)−EDTA complexes was directly immobilized onto microwell plates. The serum sample containing metallothionein(MT)-bound and non-MT-bound Cd(II) was acidified to displace the Cd(II) from MT. The sample was then treated with metal-free EDTA to convert Cd(II) to Cd(II)−EDTA complexes. A mixture of Cd(II)−EDTA complexes derived from serum samples and Cd(II)−EDTA conjugated with peroxidase enzyme was incubated in the wells to compete for binding sites of the immobilized antibody. After addition of peroxidase substrate, the bound fraction of the enzyme conjugate was measured by a microplate reader, and the signal was inversely proportional to the concentration of the Cd(II) in the sample. The assay limit of detection was 0.24 μg/L, and the effective working range at coefficient of variation of ≤10% was 0.24−100 μg/L. Analytical recovery of spiked Cd(II), in the concentration range between 0.8 and 50 μg/L, was 97.8 ± 4.0%. The assay was selective for Cd(II); other metal ions (Mn, Co, Cu, Zn, Mg, Hg, Ca, Ni, Fe, and Pb), tested at concentrations considerably higher than those present in human serum, did not significantly interfere with the assay. The assay results correlated well with those obtained by graphite furnace atomic absorption spectrometry (r = 0.984).