Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 62, Issue 27, Pages 6473-6480Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jf501382t
Keywords
fluorescence sensor; RBL-2H3 mast cells; fish parvalbumin; CD63; green fluorescent protein; confocal laser scanning microscopy
Funding
- National Research Program [2011BAK10B03]
- 973 National Basic Research Program of China [2012CB720804]
- Program for New Century Excellent Talents in Jiangnan University
- Commonwealth Project of the Ministry of Agriculture [201203069-1]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
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In this study, we developed a rat basophilic leukemia cell (RBL-2H3) fluorescence sensor to detect and identify the major fish allergen parvalbumin (PV). We constructed and transfected a CD63-enhanced green fluorescent protein (EGFP) plasmid into RBL cells through a highly efficient, lipid-mediated, DNA-transfection procedure. Stable transfectant RBL cells were then obtained for a cell fluorescence assay with confocal laser scanning microscopy. Results show that the cell surface expression of CD63 reflects degranulation, indicating that a fluorescence assay with these cells could efficiently measure the activation of antigen-stimulated transfectant cells and detect antigens with a nanogram level. Therefore, this cell-based fluorescence biosensor technique for detecting fish PV exhibits promise for quantifying fish PV after anti-PV immunoglobulin E (IgE) stimulation. Results show that fluorescence intensities increased with purified PV concentrations from 1 to 100 ng/mL, with a detection limit of 0.35 ng/mL [relative standard deviation (RSD) of 4.5%1, confirmed by beta-hexosaminidase assays. These rat basophilic leukemia (RBL) mast cells transfected with the CD63 EGFP gene and responded to PV only when they were sensitized with the specific IgE antibody. This demonstrates the utility of this highly sensitive biosensor for food allergen detection and prediction.
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