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 ...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.展开更多
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)
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＇,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.
