ORIGINAL PAPER Highly sensitive and specific determination of mercury(II) ion in water, food and cosmetic samples with an ELISA based on a novel monoclonal antibody Yuzhen Wang & Hong Yang & Michael Pschenitza & Reinhard Niessner & Yuan Li & Dietmar Knopp & Anping Deng Received: 20 February 2012 / Revised: 13 April 2012 / Accepted: 15 April 2012 / Published online: 4 May 2012 # Springer-Verlag 2012 Abstract Mercury is one of the most toxic heavy metals present in the environment. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the determination of Hg 2+ was developed. A new bi- functional ligand, 6-mercaptonicotinic acid (MNA), which contains a pyridine ring bearing a carboxylic group and a mercapto group, was selected for the preparation of immu- nogen. After immunization of mice and performing the hybridoma technique, the obtained mAb was characterized for its binding affinity and selectivity for Hg 2+ . Based on this novel mAb, an ELISA was established. At optimal experimental conditions, the standard curve of the ELISA for Hg 2+ was constructed in concentration range of 0.1– 100 ng mL -1 . The values of IC 50 and LOD of the assay were found to be 1.12 and 0.08 ng mL -1 . The cross- reactivity was lower than 2 % with MNA, CH 3 Hg, and CH 3 Hg-MNA and was 11.5 % and 4.4 % for Hg + and Au 3+ , respectively. No cross-reactivity was found with other metal ions such as Cu 2+ , Sn 2+ , Ni 2+ , Mn 2+ , Pb 2+ , Zn 2+ , Cd 2+ , Fe 2+ , Co 2+ , Mg 2+ , Ca 2+ , and anions such as Cl - , NO 3 - , NO 2 - , HCO 3 - ,F - , and SO 4 2- , indicating that the assay displays not only high sensitivity but also high selec- tivity. Different kinds of samples including water, milk, green vegetable, kelp, facial cleanser, and night cream were spiked with Hg 2+ and the extracts were analyzed by ELISA. Acceptable recovery rates of 80.0–113.0 % and coefficients of variation of 1.9–18.6 % were obtained. A linear relation- ship between ELISA and cold-vapor atomic fluorescence spectroscopy (CV-AFS) as indicated by a correlation coef- ficient of 0.97 for liquid samples (water samples) and 0.98 for other samples was obtained. The proposed mAb-based ELISA provides a feasible analytical method for highly sensitive and specific, fast, simple, and accurate determina- tion of uncomplexed trace Hg 2+ in environmental and food samples. Keywords Mercury . Enzyme-linked immunosorbent assay . Monoclonal antibody . Bifunctional ligand . 6-Mercaptonicotinic acid Introduction Heavy metal pollution of the environment is considered a worldwide problem. At least 20 metals are known to be Published in the topical collection Analytical Challenges in Environmental and Geosciences with guest editor Christian Zwiener. Y. Wang : Y. Li : A. Deng College of Chemistry, Sichuan University, Chengdu 610064, China H. Yang College of Pharmacy, Soochow University, Suzhou 215123, China M. Pschenitza : R. Niessner : D. Knopp (*) Chair for Analytical Chemistry, Institute of Hydrochemistry and Chemical Balneology, Technical University Munich, Marchioninistrasse 17, 81377 Munich, Germany e-mail: dietmar.knopp@ch.tum.de A. Deng (*) College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China e-mail: denganping6119@yahoo.com.cn A. Deng e-mail: denganping@suda.edu.cn Anal Bioanal Chem (2012) 403:2519–2528 DOI 10.1007/s00216-012-6052-1