2.4.1 Multiplex sIgE and total IgE assay
Specific IgEs against either one particular respiratory allergen or a mixture of various respiratory allergens were tested with the MSD assay. Very frequent respiratory allergens were included, individually and in mixtures, as shown in Table 1. A food allergen mixture which can have an impact on respiratory functioning was also included (i.e. the mix for nut allergens). The assay was based on the binding of specific IgEs of the serum samples to the biotinylated allergens and the binding of the total IgE of the serum samples to a biotinylated capture antibody. The biotinylated allergens and capture antibody were supplied by Radim Diagnostics.
To perform the MSD assay, a U-plex 96-well (10-spot) plate was used. Each well contains ten individual spots and each spot can be linked to a different biomarker with specific linkers (see graphical abstract). It is possible to run a partial plate, but the protocol described below states the volumes for a full plate. First, nine specific allergens (to measure sIgEs) and an anti-IgE capture antibody (to quantify total IgE) were coupled to one of the 10 different linkers by adding 200 µL of the biotinylated allergen/capture antibody to 300 µL of the chosen linker containing a streptavidin binding site. All linker-solutions were incubated for 30 minutes at room temperature while shaking. Then 200 µL stop solution (from MSD) was added to prevent further binding and incubated for another 30 minutes. Afterwards, 600 µL of all 10 linker-allergen/antibody solutions were combined into a single tube. When less than 10 spots (allergens) were used, extra stop solution was added to get a total volume of 6 mL. Next, 50 µl of this solution was pipetted into each well to coat the spots on the plate with the appropriate allergen, followed by one hour incubation. Meanwhile, the standard curve solutions were prepared by diluting a stock of total IgE (from Radim) with diluent 43 (from MSD) to: 100 IU/mL, 33.33 IU/mL, 11.11 IU/mL, 3.70 IU/mL, 1.23 IU/mL, 0.41 IU/mL, 0.10 IU/mL and 0 IU/mL. Subsequently, the plate was washed three times with PBS-tween (PBS-T; 0.05%, 150 µL per well), and 25 µL of diluent 43 was added to each well. Then 25μL of the corresponding standard series solution or serum sample, containing specific IgEs, was added to the appropriate predetermined wells and incubated overnight at 4°C. The next day, the plate was washed and 50 µL of the custom-made sulfo-tagged detection antibody (2 µg/mL, diluted with diluent 3 from MSD) was added to each well, which completed the immunoassay ‘sandwich’. This detection antibody was purchased from Radim Diagnostics and labelled with a sulfo-tag by MSD. After an hour of incubation, the plate was washed again with PBS-T and 150 µL of Read Buffer T was added to each well. Finally, the plate was read within the next 2-5 min and electrochemiluminescent (ECL) signals (at a wavelength of 620 nm) were detected with the MESO QuickPlex SQ 120 device from MSD. Using specific MSD-software, values of the ECL signals were generated as output data, which were converted to concentration values in IU/mL through calculations with the 4PL (four-parameter logistic) curve of the total IgE, where IU/mL corresponds to kU/L (Figure S1).