Indirect Competitive Immunoassay for Mercury Ion Determination Using Polyclonal Antibody Against the Hg-GSH Complex

Abstract

Hg is a highly toxic heavy metal in the environment. Detection and quantification of Hg plays an important role in the study of environmental contamination with this metal. A polyclonal antibody that recognizes Hg-ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA) was produced by immunizing the New Zealand white rabbit with Hg conjugated to bovine serum albumin (BSA) via a bifunctional chelator, glutathione (GSH), and the resulting antiserum was screened through indirect noncompetitive enzyme-linked immunosorbent assay (ELISA) using GSH-ovalbumin (OVA) and Hg-GSH-OVA. The polyclonal antibody displayed high levels for the relative Hg-GSH-OVA conjugate. The concentration of Hg2+was quantified based on the ability of its EDTA complexes to inhibit the binding of the goat anti-rabbit IgG conjugated with horseradish peroxidase and, subsequently, color formation in the assay. The indirect competitive ELISA was specific to Hg2+, with the lowest detection limit reaching 9.55 ng/mL. Cross-reactivities with other metals were less than 0.001% except for Cd2+, which showed a slight cross-reactivity in indirect competitive ELISA. The recoveries from deionized water were in the range of 90% to 102%. Results indicated that the ELISA based on polyclonal antibody could be a very promising analytical tool for rapid (3 to 5 hr) and sensitive determination of Hg and other metal ions in the environment. Additionally, the indirect competitive ELISA can potentially be applied in environmental forensics studies, as a sensitive and inexpensive monitoring method for environments polluted with Hg and possibly other metal ions.