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.