Jiang Donglei, Jiang Hui, Ji Jian, Sun Xiulan, Qian He, Zhang Genyi, Tang Lili
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.
J Agric Food Chem. 2014 Jul 9;62(27):6473-80. doi: 10.1021/jf501382t. Epub 2014 Jun 26.
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.
在本研究中，我们开发了一种大鼠嗜碱性白血病细胞（RBL - 2H3）荧光传感器，用于检测和鉴定主要的鱼类过敏原小清蛋白（PV）。我们通过高效的脂质介导的DNA转染程序，构建并将CD63增强绿色荧光蛋白（EGFP）质粒转染到RBL细胞中。然后获得稳定转染的RBL细胞，用于共聚焦激光扫描显微镜的细胞荧光测定。结果表明，CD63在细胞表面的表达反映了脱颗粒情况，这表明用这些细胞进行荧光测定可以有效地测量抗原刺激的转染细胞的活化，并检测纳克水平的抗原。因此，这种基于细胞的检测鱼类PV的荧光生物传感器技术在抗PV免疫球蛋白E（IgE）刺激后定量鱼类PV方面显示出前景。结果表明，荧光强度随着纯化的PV浓度从1到100 ng/mL增加，检测限为0.35 ng/mL [相对标准偏差（RSD）为4.5%]，β - 己糖胺酶测定法证实了这一点。这些用CD63 - EGFP基因转染的大鼠嗜碱性白血病（RBL）肥大细胞，只有在用特异性IgE抗体致敏后才对PV作出反应。这证明了这种高灵敏度生物传感器在食品过敏原检测和预测中的实用性。
