title: Development of indirect competitive fluorescence immunoassay for 2,2',4,4'-tetrabromodiphenyl ether using DNA/dye conjugate as antibody multiple labels.
abstract: 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 microg/L, with an IC50 value of 15.6 microg/L. The calculated LOD of the immunoassay is 0.73 microg/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 microg/L BDE-47, quantification by the immunoassay was 41.9 microg/L, which compared well with the standard GC-ECD method (45.7 microg/L). The developed immunoassay provides a rapid screening tool for polybrominated diphenyl ethers in environmental samples.
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environment Sciences, Chinese Academy of Sciences, Beijing 100085, China. sumioo.van@gmail.com
authors.forename: Zi-Yan
authors.initials: ZY
authors.lastname: Fan
authors.forename: Young Soo
authors.initials: YS
authors.lastname: Keum
authors.forename: Qing-Xiao
authors.initials: QX
authors.lastname: Li
authors.forename: Weilin L
authors.initials: WL
authors.lastname: Shelver
authors.forename: Liang-Hong
authors.initials: LH
authors.lastname: Guo
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D004247:DNA / Q000737:chemistry; D003959:Diamines; D019084:Fluorescent Antibody Technique, Indirect; D055768:Halogenated Diphenyl Ethers; D009930:Organic Chemicals / Q000737:chemistry; D011075:Polybrominated Biphenyls / Q000032:analysis*; D011804:Quinolines; D011817:Rabbits
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D055768:Halogenated Diphenyl Ethers; D009930:Organic Chemicals; D011075:Polybrominated Biphenyls; D011804:Quinolines; C511295:2,2',4,4'-tetrabromodiphenyl ether; C098022:SYBR Green I; D004247:DNA