2.4.2 Singleplex total IgE assay
Total IgE was also determined via a singleplex assay using a 96-well streptavidin plate (MSD). Again, the method was based on the manufacturer’s protocol,15 but adapted for IgE-measurements, using capture antibody, standards and detection antibody from Radim Diagnostics. First of all, the biotinylated anti-IgE capture antibody (from Radim Diagnostics) is diluted in diluent 100 (from MSD) to obtain a 1/17.5 ratio. The plate was washed three times with PBS-T. Then 25 µL of the solution was added to each well of the streptavidin plate, followed by incubation for an hour while shaking. Meanwhile, the standard series solutions (as described above) were prepared. After incubation, the plate was washed again and 25 µL of diluent 43 was added to each well. Subsequently, 25 µL of the corresponding standard series solution or serum sample was added to the predetermined well and incubated overnight at 4°C. The next day, the detection antibody and Read Buffer T were added (as described above) and the plate was read.