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\begin{document}

\title{Diagnostic characterization of respiratory allergies by means of a
multiplex immunoassay.}



\author[1]{Joline Millen}%
\author[1]{Ibbo Willems}%
\author[1]{Gitte Slingers}%
\author[2]{Marc Raes}%
\author[1]{Gudrun Koppen}%
\author[3,3]{Sabine Langie}%
\affil[1]{Flemish Institute for Technological Research}%
\affil[2]{Hasselt University}%
\affil[3]{Maastricht University}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Allergic sensitization is commonly assessed in patients by performing
the skin prick test (SPT) or determining specific IgE levels in blood
samples with the ImmunoCAP assay, which measures each allergen and
sample separately. This paper explores the possibility to investigate
respiratory allergies with a high throughput method, the Meso Scale
Discovery (MSD) multiplex immunoassay, measuring IgE levels in low
volumes of blood. The MSD multiplex immunoassay, developed and optimized
with standards and allergens from Radim, was validated against the SPT
and the ImmunoCAP assay. For 18 adults (15 respiratory allergy patients
and 3 controls), blood collection and the SPT were performed within the
same hour. Pearson correlations and Bland-Altman analysis showed high
comparability of the MSD multiplex immunoassay and the ImmunoCAP assay,
except for house dust mite. The sensitivity of the MSD multiplexed assay
was [?]75\% for most allergens compared to the SPT and ImmunoCAP assay.
Additionally, the specificity of the MSD multiplex immunoassay was [?]80\%
- the majority showing 100\% specificity. Only the rye allergen had a
low specificity when compared to the SPT, probably due to
cross-reactivity. The reproducibility of the MSD multiplex immunoassay,
assessed as intra- and inter-assay reproducibility and biological
variability between different sampling moments, showed significantly
high correlations (\emph{r}=0.943-1) for all tested subjects (apart from
subject 13; \emph{r}=0.65-0.99). The MSD multiplex immunoassay is a
reliable method to detect specific IgE levels against respiratory
allergens in a multiplexed and high throughput way, using blood samples
as small as from a finger prick.%
\end{abstract}%



\sloppy




\textbf{Main text (3500 words):}

\textbf{1. INTRODUCTION}

Respiratory allergies are the most common allergies worldwide, still
increasing in frequency and severity. The World Health Organization
estimated patients suffering from allergic asthma at 235 million in 2013
and the numbers for allergic rhinitis at about 400 million in 2006
{[}1,2{]}. Although the number of patients with respiratory allergy
drastically increased on a global scale, the increase is greatest in
Western countries. In 2011, the European Federation of Allergy (EFA)
reported that respiratory allergies affected around 20-30\% of the
European population. More specifically, around 68 million patients
suffered from allergic asthma and 113 million from allergic rhinitis
{[}3{]}. To emphasize the increasing prevalence, The European Academy of
Allergy and Clinical Immunology (EAACI) reported in 2015 that half the
European population will suffer from chronic allergic diseases by 2025
{[}4{]}. Alarmingly, a vast majority of individuals affected by
allergies are children and young adults {[}5{]}. Although not being
associated with high morbidity and mortality, respiratory allergy has
generated a burden on society due to both the impact on the quality of
life and the cost of treatment. A quick and precise diagnosis to
identify the causative allergens is of fundamental importance to
establish the most adequate treatment {[}6{]}.

In addition to the history and the clinical examination, looking for IgE
sensitization is an essential step in the diagnosis of complaints
compatible with allergy. Data suggests that over 50\% of allergic
patients are multi-sensitized, making diagnosis even more challenging
{[}7{]}. Traditionally, two methods are applied: \emph{in vivo} by
executing a skin prick test (SPT) or \emph{in vitro} by the detection of
sIgE in blood by using the ImmunoCAP methodology {[}8{]}. SPT is quick
and results are immediately known, but requires trained clinical
personnel to perform it. Although SPT is sensitive, minimally invasive
and modest in costs, \emph{in vitro} methods like ImmunoCAP and ELISA
have some advantages including direct quantitation, improved safety, and
the possibility of long-term storage of specimens. Notwithstanding,
the\emph{in vitro} procedures present limitations that are an impediment
for large studies and for prospective studies to be performed over
several years. For instance, measurements for each allergen and sample
has to be performed separately, which is time-consuming and is therefore
associated with higher costs and increased possibility for technical
errors. Comparing studies of the two above mentioned test methods, the
SPT seems to be more sensitive (less false-negative results), while sIgE
immunoassays seem to be more specific (less false-positive results)
{[}9,10{]}.

During the last decade, there was a gradual evolution from performing
singleplex towards multiplex immunoassays. These multiplex immunoassays
provide several advantages over singleplex immunoassays including
increased efficiency at reduced expense, lower sample volume needed
making it interesting from a pediatric standpoint, greater output
(number of markers assessed) per sample volume and higher throughput
predicting more detailed diagnostics, thereby facilitating personalized
medicine {[}11{]}. Currently, various singleplex as well as multiplex
immunoassays are available to investigate RA-associated immunological
protein markers (e.g. by Luminex, Fireplex, Meso Scale Discovery (MSD)),
but none provided an assay to assess sIgE levels in multiplex. In this
study, we utilized the MSD multiplex immunoassays considering that this
method ensures all the above-mentioned advantages, with only 25 \selectlanguage{greek}µ\selectlanguage{english}L of
serum volume required for analysis. This particular advantage cannot be
emphasized enough, since taking blood samples from children remains a
demanding technique and ethically sensitive issue. By using the MSD
multiplex immunoassay, a finger prick could be sufficient to collect
enough sample volume to conduct the test. Likewise, biomonitoring
studies could also benefit from this advantage. Additionally, the MSD
multiplex immunoassay is a highly flexible method as you can interchange
the allergen panel according to the individual needs of the patient.
Both individual allergens and allergen mixtures could be spotted on
either a complete or partial 96-well plate, making the test even more
compelling for either individual diagnosis as well as for biomonitoring
studies. Novel techniques like the ImmunoCAP Rapid Point-of-care, which
can be performed on children and adults, still require 110\selectlanguage{greek}µ\selectlanguage{english}L of sample
volume {[}12,13{]}. Moreover, this technique is not flexible as it
operates via a fixed allergen panel.

To date, validation of multiplex immunoassays for \emph{in vitro}testing
in clinical settings is limited. In this regard, this study aimed to
investigate the possibility of diagnosing respiratory allergies with the
MSD multiplex immunoassay while simultaneously comparing this method to
both an SPT and an ImmunoCAP assay. Statistical analysis demonstrated
high comparability of the MSD multiplex immunoassay and the ImmunoCAP
assay. Both tests appear reliable in terms of sensitivity and
specificity. Moreover, the reproducibility of the MSD multiplex
immunoassay was determined, including intra- and inter-assay variation
and reproducibility over time between different sampling moments. The
reproducibility parameters established significantly high Pearson
correlations. Finally, a shorter version of the protocol, including 2h
instead of overnight incubation with the samples, provides results
within 8h after blood collection.



\textbf{2. MATERIALS AND METHODS 2.1. Study population}

The study population consisted of 18 adults (12 females and 6 males; age
range 23-50 years; mean 34.8 \selectlanguage{ngerman}± 8.2 years). Three non-allergic controls
and 15 patients, who suffered from at least one respiratory allergy,
were included. Sampling was performed in August. All subjects were asked
to stop with all anti-allergic medication for at least a week before
sampling. Written informed consent was obtained from all individuals,
the study was approved by the medical ethics committee UZA-UA (EC UZA
18/11/154).

\textbf{2.2. Skin prick test (SPT)}

Droplets of allergens, histamine (positive control) and a negative
control were applied to the forearms of the subjects. In each droplet, a
small scratch was made into the skin. Skin reactions caused by the
allergens were determined after 15 minutes by a medical doctor. The size
(wheal: length and width) of redness (flare) on the skin around the
allergens and controls was measured. The allergens used in this analysis
are shown in Table 1.

\textbf{2.3. Blood analysis}

From each individual, two blood serum tubes of 10 mL were collected. One
serum tube was sent to a clinical laboratory (Labo Rigo, Genk, Belgium),
where the ImmunoCAP assay from Thermo Fisher Scientific (Waltham, MA,
USA) was performed to measure the total IgE-level and specific
IgE-levels for respiratory allergens, according to the manufacturer's
protocol. The allergens analysed in this assay are shown in Table 1. The
blood serum of the second tube was isolated, aliquoted and stored at
-80°C for later use in the MSD multiplex immunoassay.

\textbf{2.4. MSD immunoassay}

The MSD multiplex immunoassay was used to measure the total IgE as well
as specific IgE-levels. This method is based on the U-plex protocol of
MSD (Rockville,~MD, USA) {[}14{]}, but was adapted specifically for
IgE-measurements, using biotinylated allergens and IgE standards from
Radim Diagnostics (Freiburg im Breisgau, Germany).

\emph{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 \selectlanguage{greek}µ\selectlanguage{english}L of the
biotinylated allergen/capture antibody to 300 \selectlanguage{greek}µ\selectlanguage{english}L of the chosen linker
containing a streptavidin binding site. All linker-solutions were
incubated for 30 minutes at room temperature while shaking. Then 200 \selectlanguage{greek}µ\selectlanguage{english}L
stop solution (from MSD) was added to prevent further binding and
incubated for another 30 minutes. Afterwards, 600 \selectlanguage{greek}µ\selectlanguage{english}L 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 \selectlanguage{greek}µ\selectlanguage{english}l 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 \selectlanguage{greek}µ\selectlanguage{english}L per well), and 25 \selectlanguage{greek}µ\selectlanguage{english}L of diluent 43 was added to each
well. Then 25\selectlanguage{greek}μ\selectlanguage{english}L of the corresponding standard series solution or serum
sample, containing specific IgEs, was added to the appropriate
predetermined wells and incubated overnight at 4\selectlanguage{ngerman}°C. The next day, the
plate was washed and 50 \selectlanguage{greek}µ\selectlanguage{english}L of the custom-made sulfo-tagged detection
antibody (2 \selectlanguage{greek}µ\selectlanguage{english}g/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 \selectlanguage{greek}µ\selectlanguage{english}L 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).

\emph{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 \selectlanguage{greek}µ\selectlanguage{english}L
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 \selectlanguage{greek}µ\selectlanguage{english}L of diluent 43 was added
to each well. Subsequently, 25 \selectlanguage{greek}µ\selectlanguage{english}L of the corresponding standard series
solution or serum sample was added to the predetermined well and
incubated overnight at 4\selectlanguage{ngerman}°C. The next day, the detection antibody and
Read Buffer T were added (as described above) and the plate was read.

\textbf{2.5. Data and statistical analyses}

To analyse the results, descriptive statistics were performed. The
linear correlations between the results of the clinical laboratory and
the results of the MSD multiplex immunoassay were evaluated by the
Pearson correlation coefficient (\emph{r} ). This coefficient was also
used to determine the intra- and inter-assay reproducibility and the
difference in incubation time of the MSD multiplex immunoassay. To
assess the comparability of the ImmunoCAP assay and the MSD multiplex
immunoassay, a Bland-Altman analysis was performed {[}16{]}. A
Bland-Altman plot is a scatter plot of the difference between the two
methods' means (y-axis) against the mean of the two methods (x-axis).
The calculated mean difference is shown and the 95\% confidence limits
of agreement are obtained by calculating the mean difference plus or
minus two times the standard deviation of the differences. The
sensitivity and specificity of the MSD multiplex immunoassay were
determined, both in comparison to the skin prick test and the ImmunoCAP
assay of the clinical laboratory. The threshold set for a positive test
in both the MSD and ImmunoCAP assay was 0.1 kU/L. When checking for
outliers, subject 3 was eliminated from the analyses: positive scores
were observed for all MSD multiplexed measurements (all sIgE-levels
between 0.3 and 0.7 kU/L) and consistent negative scores for the other
assays. Moreover, this person reported to be diagnosed with essential
thrombocythemia, which could explain the divergent results.

\textbf{3. RESULTS \& DISCUSSION}

\textbf{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.

\textbf{3.2. Comparison of the methods}

The results of the MSD multiplex immunoassay were compared to the two
most commonly used tests: the SPT and the ImmunoCAP assay, performed by
a clinical laboratory. When an individual scored positive for the
allergy it is shown in colour in Figure 1. When redness of the skin is
observed in the SPT, it is indicated as positive. For the tests of the
clinical lab and the MSD immunoassay, a threshold of 0.10 kU/L was used;
a result above this threshold was considered positive. Both individual
allergens and mixes of allergens were tested in parallel. The SPT
measurements of subject 16 were excluded because this individual reacted
to all pricks, including the negative control, which makes the results
doubtful. This could be due to aspecific hyperreactivity of the skin.
Most results corresponded well between the three tests, although there
were some differences, as discussed later.

\textbf{3.3. Correlations and Bland-Altman analysis}

The linear correlations between the results of the clinical lab and the
results of the MSD multiplex immunoassay are presented in Table 2. The
Bland-Altman plots, which determine the comparability of these two
methods, are shown in Figure 2. For both immune-assay analyses, the
allergens of cow's milk, \emph{C. herbarum} and the mixture of fungi
were not included due to the lack of positives among the tested
individuals (none were above 0.10 kU/L) for the MSD multiplex
immunoassay (Table 2). For house dust mite no correlation was observed,
and it showed also the least comparability to the ImmunoCAP assay
according to the corresponding Bland-Altman plot. For the other
allergens, significant correlations (\emph{r} =0.829-0.978;
P\textless{}0.001) were observed as well as good agreements with the SPT
and ImmunoCAP assay, as shown in the corresponding Bland-Altman plots.

\textbf{3.4. Sensitivity and specificity}

The results of the MSD multiplex immunoassay were compared to the
results of the ImmunoCAP assay and the SPT. The sensitivity and
specificity of the MSD multiplex immunoassay were calculated (Table 3).
Overall, the sensitivity of the MSD multiplexed assay was high for most
allergens compared to the SPT and ImmunoCAP assay as references (\selectlanguage{english}[?]75\%),
however, for some sIgEs the sensitivity values were lower. As described
above, dust mite allergy was not always detected using MSD (sensitivity:
60\%). Furthermore, the sensitivity of the MSD assay detecting sIgE
against dog epithelium and mugwort was low when using the ImmunoCAP
assay as reference (20-50\%), but was very good (100\%) in comparison
with SPT. Since the SPT is still used as the golden standard in clinical
practice, the MSD multiplex immunoassay for dog and mugwort allergies
was considered sensitive. It seemed that the ImmunoCAP assay was more
sensitive to dog and mugwort allergens, which may have indicated that
the individuals were sensitized without clinical signs of allergy. For
some sIgEs, the sensitivity could not be determined, due to a lack of
positive individuals for these allergies (Figure 1). In contrast to the
sensitivity, the specificity of the MSD multiplex immunoassay was very
high (\selectlanguage{english}[?]80\% - with the majority showing 100\% specificity). When using
the data from the SPT as reference, the only sIgE with low specificity
was rye (25\%). This was probably due to cross-reactivity of IgE
antibodies against timothy grass and rye, both species of grasses of
the\emph{Poaceae} family {[}17{]}. The cross-reactivity caused a person
allergic to timothy grass, to also test positive for sIgE against rye in
the MSD multiplex immunoassay and the ImmunoCAP assay. However, the SPT
showed only an allergic reaction to timothy grass (Figure 1). A
limitation of this study was the fact that, for the ImmunoCAP assay,
only results of three mixtures of allergens were available and could be
compared, and only one mixture was available for parallel tests in the
SPT (Table 1).

In previous studies, the sensitivity and specificity of the ImmunoCAP
assay were compared to the golden standard, the SPT {[}18,19{]}. For a
select set of allergens that were analysed both in those previous
studies with the ImmunoCAP assay and with the MSD multiplex immunoassay
in the current study, similar sensitivity and specificity were observed
(Table S2). For timothy grass, cats and dogs, the MSD multiplex
immunoassay showed higher sensitivity and specificity values than the
ImmunoCAP assay when compared to the SPT. The specificity values of dust
mite and \emph{Cladosporium herbarum} were also higher for the MSD
multiplex immunoassay, however, the sensitivity of dust mite and the
specificity of rye were higher in the ImmunoCAP assay.

\textbf{3.5. Reproducibility}

\emph{3.4.1 Intra-assay variability}

To assess the intra-assay variability of the results of the MSD
multiplex immunoassay, each sample was added to the plate in duplicate,
for both measurements of individual allergens and mixes of allergens.
The correlation between the duplicate measures was calculated for all
subjects (Table 4). The results show a very stable reproducibility for
all samples (\emph{r} =0.95-1; P\textless{}0.001).

\emph{3.4.2 Inter-assay variability}

To check the variability of the same samples between two MSD multiplex
immunoassays performed on different days, correlations were calculated
for the individual allergens of serum samples from 7 individuals. The
values in Table 4 demonstrate a good inter-assay reproducibility
(\emph{r} =0.97-1; P\textless{}0.001).

\emph{3.4.3 Biological variability over time}

For six individuals, results were available of the MSD multiplex
immunoassay from a previous blood sampling and testing, in Spring
instead of Summer, one year and three months earlier (Table 4). In the
prior measurements, plasma samples were used, while in the current
tests, serum samples were analysed. However, previously analysis of
serum and plasma samples collected at the same time showed a correlation
of \emph{r} =0.92 (P\textless{}0.001), indicating that both matrices
show comparable sIgE-levels with the MSD multiplex immunoassay. The
biological variation over time was observed to be consistent (\emph{r}
=0.94-1; P\textless{}0.001). The correlation for subject 13 was,
however, lower (\emph{r} =0.65; P\textless{}0.05), which was most
probably due to seasonal effects (sampling time Spring vs. Summer).

\emph{3.4.4 Comparison incubation overnight or 2 hours}

The MSD multiplex immunoassay protocol states that after adding the
standard series and samples, either an incubation overnight at 4degC or
for 2 hours at room temperature while shaking the plate, can be applied.
To compare the two different incubation periods, the assay was performed
in both ways for the individual allergens (Table 4). Correlations were
very high (\emph{r} =0.91-1; P\textless{}0.001), meaning that both an
incubation overnight as well as for 2 hours can be applied.

\textbf{4. CONCLUSION}

The multiplex immunoassay from Meso Scale Discovery, combined with
standards and allergens from Radim, allowed high-throughput analysis of
specific IgEs against respiratory allergens in serum samples. The
results were reproducible and also comparable to the commonly used,
well-established methods (SPT and ImmunoCAP assay). The MSD multiplex
immunoassay needs small blood sample volumes and is also very versatile,
both individual allergens as well as mixes of allergens can be analysed.
The choice of allergens can be adapted easily to the patients' or
researchers' needs.

\textbf{Acknowledgements}

We thank our study volunteers for participating in this study.

SL was the beneficiary of a post-doctoral fellowship (12L5216N) provided
by The Research Foundation-Flanders (FWO) and the Flemish Institute of
Technological Research (VITO). The work was funded by an FWO Research
Grant (1523817N).

\textbf{Author contribution}

SL and GK conceived and designed the experiments. SL, IW, JM and GS
recruited volunteers and processed samples. MR performed the skin prick
test and venepuncture. JM, IW and SL performed the experiments. JM, SL,
and IW analysed the data. SL, GK, and RM contributed the
reagents/materials/analysis tools. JM, IW, and SL wrote the paper. All
authors evaluated and approved the manuscript.

\textbf{CONFLICTS OF INTEREST}

The authors declare that there are no conflicts of interest.

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\textbf{TABLES}

\textbf{Table 1.} Overview of the allergens used in the different tests.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Allergens & Skin prick test & Clinical laboratory & MSD multiplex
immunoassay\tabularnewline
\midrule
\endhead
Dust mite (d1) & X & X & X\tabularnewline
Cat (e1) & X & X & X\tabularnewline
Dog (e5) & X & X & X\tabularnewline
Cow's milk (f2) & X & X & X\tabularnewline
Rye (g12) & X & X & X\tabularnewline
Timothy grass (g6) & X & X & X\tabularnewline
\emph{Cladosporium herbarum} (m2) & X & X & X\tabularnewline
Birch (t3) & & X & X\tabularnewline
Mugwort (w6) & X & X & X\tabularnewline
Mix animals (ex1): cat, dog, horse and cow & & & X\tabularnewline
Mix nuts (fx1): peanut, walnut, hazelnut, almond and coconut & & &
X\tabularnewline
Mix grasses (gx3): sweet vernal grass, perennial ryegrass, timothy
grass, rye and common velvet grass & & X & X\tabularnewline
House mix (hx1): house dust, dust mite \emph{Dermatophagoides
pteronyssinus}, dust mite \emph{Dermatophagoides farina} and cockroach &
& & X\tabularnewline
Mix fungi (mx1): \emph{Penicillium notatum}, \emph{Cladosporium
herbarum}, \emph{Aspergillus fumigatus} and \emph{Alternaria alternate}
& & X & X\tabularnewline
Mix trees (tx2): hazel, birch, alder and ash & X & X & X\tabularnewline
Mix weeds (wx2): \emph{Alsem ambriosa}, mugwort, daisy, dandelion and
narrow-leaf plantain & & & X\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2.} Pearson correlation coefficient (\emph{r} ) and its
P-value between the results of the ImmunoCAP assay and the MSD multiplex
immunoassay.\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
& Correlation \emph{(r)}\tabularnewline
\midrule
\endhead
Dust Mite & 0.271\tabularnewline
Cat & 0.829**\tabularnewline
Dog & 0.978**\tabularnewline
Rye & 0.952**\tabularnewline
Timothy grass & 0.904**\tabularnewline
Birch & 0.954**\tabularnewline
Mugwort & 0.976**\tabularnewline
Mix grasses & 0.969**\tabularnewline
Mix trees & 0.929**\tabularnewline
\bottomrule
\end{longtable}

**P-value \textless{} 0.001 significance level.

\textbf{Table 3.} Sensitivity and specificity of the MSD multiplex
immunoassay, comparing the MSD results to the ImmunoCAP assay and SPT.
0.10 kU/L was set as threshold for a positive test of both MSD and
ImmunoCAP.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& \textbf{Sensitivity (\%)} & \textbf{Sensitivity (\%)} &
\textbf{Specificity (\%)} & \textbf{Specificity (\%)}\tabularnewline
\midrule
\endhead
& ImmunoCAP & Skin prick test & ImmunoCAP & Skin prick
test\tabularnewline
Dust mite & 60 & 60 & 100 & 100\tabularnewline
Cat & 100 & 75 & 100 & 100\tabularnewline
Dog & 20 & 100 & 100 & 100\tabularnewline
Cow's milk & NA & NA & 100 & 100\tabularnewline
Rye & 100 & NA & 100 & 25\tabularnewline
Timothy grass & 100 & 100 & 100 & 100\tabularnewline
\emph{C. herbarum} & NA & NA & 100 & 100\tabularnewline
Birch & 100 & & 80 &\tabularnewline
Mugwort & 50 & 100 & 100 & 93.3\tabularnewline
Mix grasses & 100 & & 100 &\tabularnewline
Mix fungi & NA & & 100 &\tabularnewline
Mix trees & 85.7 & 85.7 & 90 & 100\tabularnewline
\bottomrule
\end{longtable}

\emph{NA: not applicable.}

\emph{}

\textbf{Table 4.} Pearson correlation coefficients and their P-values
for comparison of results within one assay (intra-assay reproducibility)
and between assays over time (inter-assay reproducibility) as well as
for biological variability over time (two different sampling times) and
differences in incubation times (overnight vs. 2h incubation) of the MSD
multiplex immunoassay.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Subject} & \textbf{Intra-assay reproducibility} &
\textbf{Inter-assay reproducibility} & \textbf{Biological variability
over time} & \textbf{Difference in incubation time}\tabularnewline
\midrule
\endhead
1 & 1.000** & 0.998** & 0.976** & 0.958**\tabularnewline
2 & 0.996** & 1.000** & 0.994** & 1.000**\tabularnewline
3 & 0.994** & & & 0.996**\tabularnewline
4 & 0.997** & & & 1.000**\tabularnewline
5 & 0.999** & & & 0.989**\tabularnewline
6 & 1.000** & 0.997** & 0.943** & 0.995**\tabularnewline
7 & 0.990** & 1.000** & 1.000** & 1.000**\tabularnewline
8 & 0.986** & & & 0.948**\tabularnewline
9 & 0.999** & & 0.962** & 0.998**\tabularnewline
10 & 0.981** & & & 0.966**\tabularnewline
11 & 0.996** & 0.979* & & 0.993**\tabularnewline
12 & 1.000** & & & 1.000**\tabularnewline
13 & 0.997** & 0.996** & 0.652* & 0.999**\tabularnewline
14 & 0.992** & & & 0.997**\tabularnewline
15 & 0.996** & & & 0.925**\tabularnewline
16 & 0.999** & 0.970** & & 0.999**\tabularnewline
17 & 0.945** & & & 0.913**\tabularnewline
18 & 1.000** & & & 0.998**\tabularnewline
\bottomrule
\end{longtable}

\emph{**P-value \textless{} 0.001 significance level. *P-value
\textless{} 0.05 significance level.}



\textbf{FIGURE LEGENDS}

\textbf{Figure 1.} Results of three methods used to measure the allergy
(IgE) levels in N=17 individuals. Green: positive result in the skin
prick test. Yellow: positive result in the ImmunoCAP assay. Red:
positive result in the MSD multiplex immunoassay. Grey: negative result.

\textbf{Figure 2.} Bland-Altman plots of the different allergens or
mixes of allergens demonstrating the comparability of the MSD multiplex
immunoassay to the ImmunoCAP assay. The difference between the two
methods (y-axis) is plotted against the mean of the two assays (x-axis).
The two horizontal red lines represent the 95\% confidence interval of
the mean difference.

\par\null

\textbf{Hosted file}

\verb`Figure 1_Millen_CEI.pptx`  available at \url{https://authorea.com/users/319751/articles/458463-diagnostic-characterization-of-respiratory-allergies-by-means-of-a-multiplex-immunoassay}

\textbf{Hosted file}

\verb`Figure 2_Millen_CEI.pptx`  available at \url{https://authorea.com/users/319751/articles/458463-diagnostic-characterization-of-respiratory-allergies-by-means-of-a-multiplex-immunoassay}

\selectlanguage{english}
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