title: Mast-cell-based fluorescence biosensor for rapid detection of major fish allergen parvalbumin.
abstract: 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%], confirmed by β-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.
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, People's Republic of China.
authors.forename: Donglei
authors.initials: D
authors.lastname: Jiang
authors.forename: Hui
authors.initials: H
authors.lastname: Jiang
authors.forename: Jian
authors.initials: J
authors.lastname: Ji
authors.forename: Xiulan
authors.initials: X
authors.lastname: Sun
authors.forename: He
authors.initials: H
authors.lastname: Qian
authors.forename: Genyi
authors.initials: G
authors.lastname: Zhang
authors.forename: Lili
authors.initials: L
authors.lastname: Tang
mesh_terms: D000485:Allergens / Q000032:analysis* / Q000276:immunology; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D005399:Fishes; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008407:Mast Cells / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D010320:Parvalbumins / Q000032:analysis* / Q000276:immunology; D010957:Plasmids / Q000235:genetics; D051381:Rats; D060149:Tetraspanin 30 / Q000235:genetics / Q000276:immunology; D014162:Transfection
chemical_list: D000485:Allergens; D010320:Parvalbumins; D060149:Tetraspanin 30; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins