Label-free homogeneous FRET immunoassay for the detection of mycotoxins that utilizes quenching of the intrinsic fluorescence of antibodies

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Title
Label-free homogeneous FRET immunoassay for the detection of mycotoxins that utilizes quenching of the intrinsic fluorescence of antibodies
Author(s)
T Li; Ju Young Byun; Bo Bae Kim; Yong Beom Shin; M G Kim
Bibliographic Citation
Biosensors & Bioelectronics, vol. 42, no. 1, pp. 403-408
Publication Year
2013
Abstract
The phenomenon of fluorescence quenching of an antibody by a specific ligand was applied in developing a technique for detection of mycotoxins, such as aflatoxin B1 (AFB1), ochratoxin A, and zearalenone. Studies showed that the intrinsic fluorescence of tryptophan (Trp) residues in antibodies, promoted by excitation at 280nm, is quenched upon binding of specific mycotoxin ligands. Fluorescence quenching in FRET system takes place in these systems as a consequence of the overlap of the emission spectra of antibody donors with the absorption spectra of the mycotoxins. Further studies focusing on the detection of AFB1 revealed that the Fab fragment, the variable region of the antibody where specific binding of AFB1 occurs, can be utilized to increase the sensitivity of the detection system. The results demonstrate that fluorescence of the Fab fragment is almost completely quenched by AFB1 whereas emission from intact anti-AFB1 is only partially quenched by this mycotoxin. The limits of detection (LODs) were found to be 0.85 and 0.09ngmL-1 for assays using the intact antibody and the Fab fragment, respectively, corresponding to a 10-fold enhancement. A practical application of the Fab fragment based assay system was demonstrated by its use in the detection of AFB1 in spiked barley grain samples. The observations made in this effort show that the new, label-free, non-competitive, and homogeneous FRET immunoassay strategy, which requires a simple sample preparation procedure, serves as a powerful tool for the rapid and sensitive quantitative determination of organic substances such as mycotoxin.
Keyword
AntibodyFab fragmentFRET immunoassayHomogeneousLabel-freeMycotoxin
ISSN
0956-5663
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/j.bios.2012.10.085
Type
Article
Appears in Collections:
Division of Biomaterials Research > Bionanotechnology Research Center > 1. Journal Articles
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