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.