Environmental analytical methods

Development of indirect competitive fluorescence immunoassay for 2,2′,4,4′-tetrabromodiphenyl ether using DNA/dye conjugate as antibody multiple labels

Zi-Yan Fan , Young Soo Keum , Qing-Xiao Li , Weilin L. Shelver , Liang-Hong Guo


Received September 29, 2011,Revised November 30, 2011, Accepted , Available online July 08, 2012

Volume 24,2012,Pages 1334-1340

An indirect competitive fluorescence immunoassay using a DNA/dye conjugate as antibody multiple labels was developed on 96-well plates for the identification and quantification of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) in aqueous samples. A hapten, 2,4,2′-tribromodiphenyl ether-4′-aldehyde, was synthesized, and was conjugated to bovine serum albumin to form a coating antigen. Specific recognition of the antigen by anti-PBDE antiserum was confirmed by a surface plasmon resonance measurement. In the immunoassay, the coating antigen was adsorbed on a 96-well plate first, and a sample, antiserum and biotinylated goat anti-rabbit secondary antibody were then added and reacted sequentially. A biotinylated, double-stranded DNA with 219 base pairs was attached to the secondary antibody by using streptavidin as a molecular bridge. In situ multiple labeling of the antibody was accomplished after addition of a DNA-binding fluorescent dye, SYBR Green I. The working range of the immunoassay for the BDE-47 standard was 3.1-390 μg/L, with an IC50 value of 15.6 μg/L. The calculated LOD of the immunoassay is 0.73 μg/L. The immunoassay demonstrated relatively high selectivity for BDE-47, showing very low cross-reactivity (< 3%) with BDE-15, BDE-153 and BDE-209. With a spiked river water sample containing 50 μup g/L BDE-47, quantification by the immunoassay was 41.9 μg/L, which compared well with the standard GC-ECD method (45.7 μg/L). The developed immunoassay provides a rapid screening tool for polybrominated diphenyl ethers in environmental samples.

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