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.