Wan Daniel, Alcocer Marcos J C, Falcone Franco H
School of Pharmacy, University of Nottingham, Nottingham, UK.
Division of Food Sciences, School of Biosciences, University of Nottingham, Loughborough, UK.
Methods Mol Biol. 2025;2929:153-162. doi: 10.1007/978-1-0716-4595-6_12.
We have established a workflow allowing printing of allergens and control proteins into the wells of a MaxiSorp 96-well plate. Allergens are printed e.g., in a 4 × 4 pattern into each well of the plate. Prior to adding to the printed plates, a humanized rat basophil leukemia (RBL)-derived fluorescent reporter cell line (e.g., RBL NFAT DsRed) is sensitized overnight with the IgE-containing serum to be tested. Next, the humanized reporter cell line is added to the wells with the printed allergens and incubated for 24 h in a cell standard culture incubator. If the serum sample contains enough IgE specific for the printed allergens, NFAT-dependent signal transduction will occur, leading to synthesis of intracytosolic DsRed protein during an overnight incubation. Fluorescence can then be measured using a microplate reader using the well scan function.
我们已经建立了一种工作流程，可将过敏原和对照蛋白打印到MaxiSorp 96孔板的孔中。例如，将过敏原以4×4的模式打印到板的每个孔中。在添加到打印好的板之前，用待测试的含IgE血清对人源化大鼠嗜碱性粒细胞白血病（RBL）衍生的荧光报告细胞系（例如RBL NFAT DsRed）进行过夜致敏。接下来，将人源化报告细胞系添加到含有打印过敏原的孔中，并在细胞标准培养箱中孵育24小时。如果血清样品含有足够的针对打印过敏原的IgE，则会发生NFAT依赖性信号转导，导致在过夜孵育期间合成胞内DsRed蛋白。然后可以使用酶标仪的孔扫描功能测量荧光。
