Development of indirect competitive fluorescence immunoassay for 2,2¡ä,4,4¡ä-tetrabromodiphenyl ether using DNA/dye conjugate as antibody multiple labels
详细信息 查看全文
- 作者：Zi-Yan Fan ; Young Soo Keum ; Qing-Xiao Li ; Weilin L. Shelver ; Liang-Hong Guo
- 关键词：polybrominated diphenyl ethers ; fluorescence immunoassay ; microplate ; multiple labeling
- 刊名：Journal of Environmental Sciences
- 出版年：2012
- 出版时间：July, 2012
- 年：2012
- 卷：24
- 期：7
- 页码：1334-1340
- 全文大小：175 K
文摘
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¡ä,4,4¡ä-tetrabromodiphenyl ether (BDE-47) in aqueous samples. A hapten, 2,4,2¡ä-tribromodiphenyl ether-4¡ä-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 ¦Ìg/L, with an IC50 value of 15.6 ¦Ìg/L. The calculated LOD of the immunoassay is 0.73 ¦Ìg/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 ¦Ìg/L BDE-47, quantification by the immunoassay was 41.9 ¦Ìg/L, which compared well with the standard GC-ECD method (45.7 ¦Ìg/L). The developed immunoassay provides a rapid screening tool for polybrominated diphenyl ethers in environmental samples.