3.1 Total IgE
As mentioned in the Materials and Methods, one of the 10 spots in the MSD multiplex immunoassays was used to detect total IgE levels, which was essential to construct a standard curve. At the same time, this spot also detected total IgE levels in the wells containing individual serum samples. However, total IgE levels assessed for the individual samples in the multiplex assay were always lower compared to the ImmunoCAP measurements. An explanation for this is the range of the standard curve, which in the multiplex MSD assay went up to 100 kU/L. The total IgE levels were above this standard curve, resulting in underestimated concentrations (Table S1) when using the 4PL curve for conversion (Figure S1). As such, one could argue to omit this spot (to detect total IgE) from the wells in which serum samples are assessed. However, when this spot was left empty, it resulted in divergent levels of sIgE against the allergens in that same well, probably due to altered chemistry of the assay in those wells compared to the wells that quantified the standard curve and thus contained 10 ‘active’ spots. Alternatively, serum samples were diluted 10x, which resulted in total IgE levels similar to ImmunoCAP, but sIgE levels against the allergens ended up below the detection limit in most cases. Diluting the samples 10x and analysing them in a parallel singleplex immunoassay allowed more accurate quantification of total IgE. These data indicate that 10x dilution of the samples is not an option for the MSD multiplex immunoassay to assess sIgE, and total IgE could not be measured accurately in the same well and is thus best analysed in a singleplex immunoassay. However, in clinical practice total IgE levels are only minimally taken into account when characterising specific allergies. Therefore, total IgE levels are not necessary to be determined in case one is interested in sIgE levels to detect specific (respiratory) allergies. The rest of this paper will thus focus on the sIgE levels for the comparison of the various methods.