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.

Keywords:

• mercury
• antibody
• enzyme-linked immunosorbent assay (ELISA)
• detection

Acknowledgement

This work was supported by the National High Technology Research and Development Program (“863” Program) of China (No. 2007AA10Z441), Science Foundation of Shanghai (No. 08DZ1900404), Special Fund for Agro-scientific Research in the Public Interest of China (No. 200903056), Science Foundation of Chongming of Shanghai (No. 10DZ1960103), and the National Natural Science Foundation of China (No. 31071860).