|FAN, ZIYAN - CHINESE ACADEMY OF SCIENCES|
|KEUM, YOUNG - KONKUK UNIVERSITY|
|LI, QING - UNIVERSITY OF HAWAII|
|GUO, LIANG-HONG - CHINESE ACADEMY OF SCIENCES|
Submitted to: Journal of Environmental Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/14/2011
Publication Date: 8/1/2012
Citation: Fan, Z.Y., Keum, Y.S., Li, Q.X., Shelver, W.L., Guo, L.H. 2012. Development of indirect competitive fluorescence immunoassay for 2,2',4,4'-tetrabromodiphenyl ether using DNA/dye conjugate as antibody multiple labels. Journal of Environmental Science. 24(7):1334-1340.
Interpretive Summary: Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants which have been widely used in many applications such as building materials, electronic products, plastic, and textiles. As a result of widespread usage as well as high stability and bioaccumulation, PBDEs were found in almost all environmental media and biological samples. A double-stranded DNA was first attached to the antibody. Fluorescent dyes were then allowed to bind to the DNA, forming a dye/DNA conjugate. Because the dye can bind to DNA at high ratios, the antibody is labeled with a large number of fluorescent dyes through the DNA. Using this method, fluorescence immunoassays on 96-well plates showed a detection limit 200 fold lower than the assay using the conventionally labeled antibodies. In the current work, the dye/DNA conjugate has been employed in the competitive fluorescence immunoassays for PBDEs. Unlike ELISA, this format is compatible with the current biochip technology, and thus can be readily developed into immunoassay biochips for the simultaneous detection of multiple analytes in a single sample.
Technical Abstract: An indirect competitive fluorescence immunoassay using 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 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 DNA-binding fluorescent dye, SYBR Green I. The working range of the immunoassay for the BDE-47 standard was 3.1 - 390 ug/L, with an IC50 value of 15.6 ug/L. The calculated LOD of the immunoassay is 0.73 ug/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 50 ug/L BDE-47 spiked river water sample, quantification by the immunoassay was 41.9 ug/L, which compared well with the standard GC-ECD method (45.7 ug/L). The developed immunoassay provides a rapid screening tool for polybrominated diphenyl ethers in environmental samples.