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

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

机构地区：[1]State Key Laboratory of Environmental Chemistry and Ecotoxicology,Research Center for Eco-environment Sciences,Chinese Academy of Sciences,Beijing 100085,China [2]College of Life and Environmental Sciences,Konkuk University in Korea,1 Hwayang-dong,Gwangjin-gu,Seoul 143-701,Korea [3]Department of Molecular Bioscience and Bioengineering,University of Hawaii at Manoa,1955 East-West Road,Honolulu,HI 96822,USA [4]USDA-ARS Bioscience Research Laboratory,1605 Albrechht Boulevard,Fargo,ND 58105,USA

出　　处：《Journal of Environmental Sciences》2012年第7期1334-1340,共7页环境科学学报（英文版）

基　　金：supported by the Chinese Academy of Sciences (No. KSSCX2-YW-G-059);the National Hi-Tech Research and Development Program of China (No.2007AA06A407);the National Natural Science Foundation of China (No. 20825519, 20890112, 20921063)

摘　　要：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 ~tg/L, with an IC50 value of 15.6 Ixg/L. The calculated LOD of the immunoassay is 0.73 Ixg/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 Izg/L BDE-47, quantification by the immunoassay was 41.9 ~tg/L, which compared well with the standard GC-ECD method （45.7 Ixg/L）. The developed immunoassay provides a rapid screening tool for polybrominated diphenyl ethers in environmental samples.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 ~tg/L, with an IC50 value of 15.6 Ixg/L. The calculated LOD of the immunoassay is 0.73 Ixg/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 Izg/L BDE-47, quantification by the immunoassay was 41.9 ~tg/L, which compared well with the standard GC-ECD method （45.7 Ixg/L）. The developed immunoassay provides a rapid screening tool for polybrominated diphenyl ethers in environmental samples.

关 键 词：polybrominated diphenyl ethers fluorescence immunoassay MICROPLATE multiple labeling 

分 类 号：Q523[生物学—生物化学] O657.3[理学—分析化学]