Deteccion especifica de ig e con microarray

A Protein Allergen Microarray Detects Specific IgE to
Pollen Surface, Cytoplasmic, and Commercial Allergen
Extracts
Katinka A. Vigh-Conrad1*¤a, Donald F. Conrad1¤b, Daphne Preuss2¤c
1 Department of Human Genetics, The University of Chicago, Chicago, Illinois, United States of America, 2 Department of Molecular Genetics and Cell Biology, The
University of Chicago, Chicago, Illinois, United States of America

Abstract
Background: Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive,
high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that
lacks pollen surface materials that may contain allergens.
Methodology/Principal Findings: We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in
sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do
this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species,
commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic
fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with
,25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a highthroughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure
allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the
phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past,
contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method
were comparable to commercial extracts, many individual allergens showed significant differences.
Conclusions/Significance: These results support the hypothesis that protein microarray technology is a useful tool for both
research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests,
making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over
time, and facilitate genetic studies on pollen allergy.
Citation: Vigh-Conrad KA, Conrad DF, Preuss D (2010) A Protein Allergen Microarray Detects Specific IgE to Pollen Surface, Cytoplasmic, and Commercial Allergen
Extracts. PLoS ONE 5(4): e10174. doi:10.1371/journal.pone.0010174
Editor: Hany A. El-Shemy, Cairo University, Egypt
Received September 1, 2009; Accepted March 22, 2010; Published April 16, 2010
Copyright: ß 2010 Vigh-Conrad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was funded by the Strategic Program for Asthma Research (www.spar-aaf.org). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: kvigh@uchicago.edu
¤a Current address: Abcam plc, Cambridge, United Kingdom
¤b Current address: Wellcome Trust Sanger Institute, Hinxton, United Kingdom
¤c Current address: Chromatin Inc., Chicago, Illinois, United States of America

development of safe and effective immunotherapy treatments [17].
According to the International Union of Immunological Societies
Allergen Nomenclature Sub-Committee, over 600 allergens have
been identified to date (http://www.allergen.org). Most of these
were identified by immunoblotting soluble allergen extracts
separated by electrophoresis with patient sera or monoclonal
antibodies [18]. Allergens identified in these studies are typically
10–70 kD cytoplasmic proteins and have diverse biological
functions [2,18,19]. Despite current successes, it remains important to continue the search for new allergens, especially from the
pollen surface, which has been largely overlooked in the past.
The pollen extracellular matrix plays an essential role in plant
reproduction [20] and numerous studies have focused on purifying
and characterizing many pollen surface materials [21]. While
some researchers have suggested these materials also play a role in

Introduction
Allergy affects 10–40% of the population [1] and results in
elevated IgE [2] - a condition that is often diagnosed with skin
prick tests (SPT) that can cause discomfort, risk anaphylaxis [3]
and can increase patient sensitivity to allergens [4]. Safer and more
quantitative alternatives for measuring allergen-specific IgE
include enzyme linked immunosorbent assay (ELISA) and
radioallergosorbent test (RAST) [5], yet their prohibitive cost
limits use to select allergens and to severely affected patients.
Microarrays potentially provide a more affordable diagnostic, and
recent studies, most of which focus on recombinant allergens, have
demonstrated their analytical and clinical feasibility [4,6–16].
Considerable effort has been invested in identifying allergens,
with the goal of providing more accurate patient diagnosis and the
PLoS ONE | www.plosone.org

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April 2010 | Volume 5 | Issue 4 | e10174

Pollen Allergen Microarray

allergy [22–25], in most cases, the allergenic role of the pollen
surface has not been explored. This is perhaps because
commercial suppliers of pollen extracts used for research (e.g.
identification of allergens), as well as diagnostics (e.g. SPT and
RAST) and treatment (e.g. immunotherapy), wash pollen with
organic solvents such as ether and acetone before extraction to
remove possible contaminants, including microbes [26]. This strips
away molecules from the pollen surface - a multilayered structure
comprised of an internal cellulose layer (intine), an outer (exine)
wall, and an extracellular matrix (the pollen coat) containing
lipophilic proteins, lipids, and small molecules [27,28]. Pollen coat
components are primarily synthesized by anther cells that
surround developing pollen grains [29], not the pollen itself [30].
Consequently, whether pollen allergens are identified by immunoblotting soluble protein extracts from commercially washed
pollen [31,32] or by screening expression libraries derived from
pollen cDNAs with patient sera [33], pollen surface materials are
likely to be overlooked. This is likely to account for the observation
that many previously identified pollen allergens can be localized to
the pollen cytoplasm [2,19].
Here, we separately examined extracts of the pollen surface and
cytoplasm from a wide range of plant species, taking care to collect
material from unwashed pollen grains and to solubilize lipophilic
pollen fractions. These extracts were arrayed along with
commercially available allergens in microarray format that can
detect allergen-specific IgE using only small amounts (,25 uL) of
sera. We screened sera from 176 individuals with elevated total
IgE and demonstrated that the allergen microarray is a
reproducible measure of allergen-specific IgE. This assay provides
extensive allergen sensitization information while using only
limited resources, supporting the hypothesis that allergen microarray technology is a more efficient and economically feasible
diagnostic that currently used approaches.

Table 1. Allergen list.

Category

Common name

Grass pollen Bermuda grass

Scientific name

Abbr.
Ber

Poa pratensis

Blu

Johnson grass

Sorghum halepense

Jhn

Orchard grass

Dactylis glomerata

Orc

Ryegrass, Perennial

Lolium perenne

Rye

Timothy grass

Phleum pratense

Tim

Artemisia ambrosioides

Mug

Ragweed, Short

Ambrosia artemisiifolia

Rag

Alder, European

Alnus glutinosa

Ald

Ash, White

Fraxinus americana

Ash

Birch, White

Betula populifolia

Bir

Cedar, Mountain

Juniperus ashei

Ced

Cottonwood, Eastern

Populus deltoides

Cot

Elder, Box

Acer negundo

Eld

Elm, American

Ulmus americana

Elm

Mulberry, Red

Morus rubra

Mul

Oak, Red

Quercus rubra

Rok

Oak, White

Quercus alba

Wok

Olive

Olea europaea

Olv

Pecan

Carya illinoensis

Pec

Sycamore, Western

Platanus racemosa

Syc

Walnut, Black

Juglans nigra

Wal

Alternaria

Aspergillus niger

Asp n

Aspergillus

Alternaria alternata

Alt a

Dustmite, American

Dermatophagoides farinae

Der f

Dustmine, European

Dermatophagoides pteronyssinus Der p

Cockroach, American

Periplaneta americana

Acr

Cockroach, German

Blattella germanica

Gcr

Cat hair

Felis catus

Cat

Dog epithelia

Canis familiaris

Dog

Dust

NA

Dst

Weed pollen Mugwort, Common

Tree pollen

Non-pollen

Materials and Methods
Ethics Statement
This research was approved by the University of Chicago IRB
and documentation of this approval is available from their office
under protocol #15100B. Sera from patients were de-identified
and data was analyzed anonymously.

Abbr. – abbreviations.
doi:10.1371/journal.pone.0010174.t001

MIAME compliance and data availability
The microarray experiments described in this manuscript are
MIAME compliant and the raw data has been deposited in
ArrayExpress (http://www.ebi.ac.uk/microarray-as/ae/); accession number E-MTAB-177.

To isolate the pollen cytoplasm, washed pollen (from previous
step) was hydrated with TBS and mechanically pulverized with sea
sand (Fisher, Pittsburgh, PA). After separation by filtration through
a glass filter, proteins were precipitated with 80% ice-cold acetone
and pellets were washed until white.
Pellets were resuspended in suspension buffer (50% Protein
Printing Buffer (ArrayIt, Sunnyvale, CA), 25% TBS-T, 10%
glycerol). All protein solutions, including the commercial allergen
extracts, were quantified with BCA Protein Assay Kit according to
manufacturer’s protocol (Pierce, Rockford, IL) and diluted to a
concentration of 1 ug/uL in suspension buffer.

Allergen extract preparation
Raw un-defatted pollen from 22 allergenic plant species,
allergen extracts of the same pollens, and allergen extracts of
non-pollen allergens were purchased from Greer (Lenoir, NC). For
a complete list, including full names, scientific names, and
abbreviations, see Table 1. Recombinant Amb a 1, Bet v 1, Phl p
2, Alt a 1, and Der p 1 were purchased from Indoor Biotechnologies
Inc. (Charlottesville, VA).
To isolate pollen surface materials, raw un-defatted pollen was
suspended in cyclohexane and vortexed. After separation by
filtration through a glass filter, the cyclohexane was evaporated
and the remaining material was resuspended in TBS-T (150 mM
sodium chloride, 10 mM Tris base, pH 7.4, 1% Tween-20).
Proteins were precipitated with 80% ice-cold acetone and the
pellets were washed until white.

Cynodon dactylon

Bluegrass

Serum samples
Sera from de-identified individuals with high levels of total IgE
(.300 kU/L) and pooled sera from 500 randomly selected
individuals were purchased from Bioreclamation Inc. (East
Meadow, NY). Additional sera from 76 de-identified patients
were obtained from the University of Chicago in vitro allergy
laboratory.
2



total of 80 allergens printed in triplicate: pollen surface and
cytoplasmic protein fractions and commercial extracts from the
pollen of six grasses, two weeds, and 13 trees; commercial extracts
of cat hair, dog epithelia, dust, two mites, two cockroaches, and
two molds; and five recombinant major allergens. Negative
controls (human serum albumin (HSA) and buffer) and standard
calibration curves of purified IgE (Fitzgerald), IgG, and IgA
(Bethyl) at 200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 pg/spot
were printed in six replicates. Microarrays were stored at room
temperature in vacuum-sealed boxes until use.

SDS-PAGE and dot blots
Commercial allergen extracts and cytoplasmic and surface
protein fractions were separated by single dimension SDS-PAGE
using 5% stacking and 12.5% separating gels. Gels were stained
with Coomassie blue according to standard protocols.
For the dot blots, 1 uL of pollen fractions were spotted onto
nitrocellulose membranes at a concentration of 2 ug/uL. Dried
membranes were washed, blocked with PBS-T containing 5% dry
milk, and incubated with 20% pooled human sera overnight at
4uC followed by human anti-IgE secondary antibody conjugated
with horseradish peroxidase (HRP) (Serotec, Raleigh, NC). Bound
antibodies were detected using SuperSignal West Pico Chemiluminescent kit using manufacturers’ protocol (Pierce, Rockford, IL).
Resulting chemiluminescence was detected on Kodak BioMax
XAR film (Fisher, Pittsburgh, PA). Washing with PBS-T was done
between all steps.

Allergen microarray immunoassay
Slides containing allergen arrays were washed with PBS-T,
rinsed with ddH2O, blocked for 120 minutes at room temperature
with BlockIt buffer (ArrayIt, Sunnyvale, CA), rinsed with ddH2O,
and washed with PBS-T. Gaskets (Grace BioLabs, Bend, OR)
were attached to the slides to create a barrier between arrays, and
a total of 50 uL of serum diluted to 25% in PBS-T containing 1%
HSA (HSA, Sigma, St. Louis, MO) was applied to the individual
reaction wells, which were then sealed to prevent evaporation.
After incubation with sera overnight at 4uC, the slides were
washed with PBS-T and rinsed with ddH2O. To detect bound IgE
antibodies, the slides were incubated for 120 minutes at 37uC with
anti-human IgE labeled with Alexa Fluor 647 (Molecular Probes,
Carlsbad, CA) diluted 1:100. Subsequently, slides were washed
with PBS-T followed by PBS, rinsed with ddH2O, spin-dried, and
stored in the dark until scanning.

ImmunoCAP RAST
ImmunoCAP RAST data from allergic patients were obtained
from the University of Chicago in vitro allergy laboratory. The
allergens tested included D. pteronyssinus, D. farinae, German
cockroach, Cat epithelium and dander, Dog epithelium, Aspergillus fumigatus, A. alternaria, Meadow fescue, Timothy, Elder,
Maple leaf sycamore, Cottonwood, White ash, Cedar, White Oak,
Lamb’s quarters, Common pigweed, Rough marshelder, Sheep
sorrel, Ribwort, and (giant) Ragweed.

Allergen-specific IgE ELISA

Scanning, quality control, and data processing

Sera were screened by ELISA for specific IgE to commercial
extracts and cytoplasmic protein fractions from six pollens
(bermuda grass, Timothy grass, mugwort, ragweed, birch, and
cottonwood). Maxisorb plates (NUNC, Rochester, NY) were
coated with allergens at a concentration of 5 ug/mL in PBS and
wells reserved for standards were coated with anti-human IgE
(KPL, Gaithersburg, MD) at a concentration of 2 ug/mL in PBS.
Plates were incubated overnight at 4uC. Plates were then washed
three times with PBS-0.05% Tween 20, blocked for 120 minutes
with 10% fetal bovine serum (FBS) in PBS at room temperature,
and then washed three times with PBS-0.05% Tween 20. A
standard serial dilution (20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and
0 ng/mL) of purified human IgE (Fitzgerald, Concord, MA) was
added to wells reserved for standards, serum samples were added
to the rest of the plate at a 1:4 dilution in 10% FBS in PBS and
incubated overnight at 4uC. Plates were then washed three times
with PBS-0.05% Tween 20, followed by incubation with mouse
anti-human IgE conjugated to HRP (Serotec, Raleigh, NC) at a
concentration of 1 ug/mL for 60 minutes a room temperature.
After washing the plates four times with PBS-0.05% Tween 20,
SureBlue TMB 1-Component Microwell Peroxidase Substrate
(KPL, Gaithersburg, MD) was added according to manufacturers’
protocol and the reaction was stopped with TMB stop solution
(KPL, Gaithersburg, MD) after sufficient color development.
Adsorbance was read at 450 nm on a plate reader and the amount
of antibody binding was extrapolated from the standard
calibration curves. Positive signal cutoff for ELISA was signals
.0.35 kU/L and positive signal cutoff for microarrays was signals
greater than the allergen specific threshold calculated from mock
arrays.

Slides were scanned using an Axon GenePix 4000B scanner
(Sunnyvale, CA) and images were analyzed using GenePix Pro 6.0
software to obtain median foreground intensity values for both red
(635 nm) and green (532 nm) channels. Irregularly shaped,
smeared, and missing spots were flagged. An automated data
processing program was developed in R version 2.5.0 (available at
http://www.r-project.org) to 1) discard flagged spots, 2) log 2
transform data, 3) correct for autofluorescence of spots, and 4)
determine allergen-specific thresholds. The IgE standard calibration curves were used to make sure that the secondary antibodies
were property working but were not used for normalization or
extrapolation of the amount of IgE binding to allergen spots
because this introduced unacceptable error into our calculations.
Autofluorescence in both the red and green channels, which
varied from spot-to-spot and allergen-to-allergen, was corrected
using the assumption that for each spot, the red channel intensity
(R) is the sum of autofluorescence (RAF) and the fluorescence of the
bound secondary antibody (RIgE). The green channel intensity (G)
was not affected by the binding of the secondary antibody (data
not shown), and was entirely the result of autofluorescence (GAF).
We observed a linear relationship between red and green channel
fluorescence on slides incubated with buffer alone (Fig. 1a), thus
RAF = mGAF+b, where b is an experimentally determined constant.
We estimated RAF from GAF on arrays containing sera by applying
linear models for each allergen separately, and this value was then
subtracted from R to obtain RIgE. When mock slides were
processed in this manner, the distribution of signal intensities
was tightly centered at 0 (Fig. 1b).
To avoid an excess of false positive signals, allergen-specific
thresholds were determined as the value at the 99th percentile from
the distribution of corrected fluorescence intensity values on mock
arrays for each allergen. These values were then subtracted from
corresponding values on sample arrays, resulting in a 1% false
positive rate. Finally, allergens with .50% missing data were
discarded and results were recorded in microarray units

Allergen microarray fabrication
Solubilized protein fractions were printed in microarray format
with 12 microarrays per standard SuperEpoxi glass slide (using
ArrayIt Stealth Printing technology). Microarrays contained a

3



Analysis of Variance (ANOVA) and correlation analyses
between pair-wise replicates were carried out using standard
functions in R. To compare the relative reactivity to different
pollen fractions, we used t-tests for matched pairs, testing the null
hypothesis of no difference between the means. Two tailed pvalues are reported.
Hierarchical clustering analysis of allergen-specific IgE was
performed in R version 2.5.0 using package pvclust [34] with the
following parameters: agglomerative method - average, distance
measure - correlation, number of bootstrap replicates - 1000.
pvclust provides two types of p-values: AU (Approximately
Unbiased) p-value, computed by multiscale bootstrap resampling
and BP (Bootstrap Probability) value, computed by normal
bootstrap resampling.

Results
Allergenicity of different types of pollen fractions
To fractionate pollen into its different components, we isolated
pollen surface materials by extraction into cyclohexane [28] and
obtained cytoplasmic fractions by the pulverization of pollen,
followed by acetone precipitation of proteins. Precipitated proteins
were solubilized and, in addition to commercial allergen extracts,
visualized on Coomassie-stained SDS-PAGE. For three different

Figure 1. Autofluorescence correction of fluorescence intensity
values. A) Scatter plot of green vs. red channel data of uncorrected
(grey circles) and autofluorescence-corrected (black circles) fluorescence intensity values. B) Histogram of autofluorescence-corrected
fluorescence intensity values on 36 mock arrays probed with secondary
antibody only (no sera).
doi:10.1371/journal.pone.0010174.g001

corresponding to the Tukey mean of corrected fluorescence
intensity of replicates.

Statistical Analyses
Coefficient of variances (CV) were calculated as the standard
deviation divided by the mean of replicates. To determine intra-slide
reproducibility, individual sera were tested on six different arrays on
the same slide. Inter-slide variation was measured by testing these sera
on three different slides on the same day. Inter-assay reproducibility
was determined by testing the same sera on three different days.
Median CV of six individuals tested for 80 allergens each where the
mean of the replicates was .0.5 were reported to avoid skews to the
data when there were small differences among low values.

Figure 2. Comparison of surface fractions, cytoplasmic fractions, and commercial allergen extracts. A) SDS-PAGE gels stained
with Coomassie blue. B) Nitrocellulose dot blots probed with sera
pooled from 500 individuals and HRP-conjugated anti-IgE.

4



Figure 3. IgE profiling using protein microarrays. A) General layout of the allergen microarray, B) scanned image of an array probed only with
secondary antibody to show autofluorescence, C–F) and scanned images of arrays probed with sera from four individuals showing different allergen
sensitization profiles. Images were pseudocolored with a color spectrum adjusted so that blue indicates low signal and red indicates high signal.

cially purchased pollen extracts (Fig. 2a). The differences in the
content of the commercial and cytoplasmic extracts, which were
both derived from washed pollen, are somewhat surprising, and

species examined (Bermuda grass, ragweed and pecan) the extracts
prepared from the pollen surface and cytoplasm contained distinct
bands, with numerous proteins that were absent from commer-

Figure 4. Serum dilution vs. fluorescence intensity values. Two serum samples, one with moderate levels of allergen-specific IgE (7746) and
one with high levels of allergen-specific IgE (7855) were tested at five different dilutions; 50%, 25%, 12.5%, 6.25%, and 3.125%. Specific IgE in
microarray units to the three fractions (surface, cytoplasm, and commercial extract) of 12 pollen allergens is shown.


5



may be attributable to different extraction methods, protein
solubilization, or protein stability.
Next, we tested whether the pollen fractions contain candidate
allergens by spotting equivalent extract quantities (normalized to
total protein) onto nitrocellulose membranes and blotting with
human sera pooled from 500 American individuals of diverse age
and ethnicity. Specific IgE binding, as detected by an anti-IgE
monoclonal antibody, was observed with each of the seven pollen
species tested, including strong signals to four pollen surface
fractions (Bermuda grass, mugwort, olive, and pecan), six
cytoplasmic fractions (Bermuda grass, ryegrass, mugwort, ragweed, pecan, and cedar), and two commercial extracts (Bermuda
grass and ryegrass) (Fig. 2b). Further characterization of allergens
contained within IgE-reactive surface fractions is described
elsewhere (manuscript in preparation).

Table 2. ANOVA of replicates.

SS

df

% SST

Array

51.191

11

0.12%

Slide

363

8

0.85%

Serum

21378

5

50.09%

Allergen

7132

78

16.71%

Error

13758

25155

32.23%

Analysis of variance statistics (ANOVA) of fluorescence intensity values; SS, sum
of squares; df, degrees of freedom; SST, percent of the total sum of squares.

Development and validation of the allergen microarray
To make a high-throughput assay testing allergen-specific IgE in
sera, microarrays were printed containing protein fractions
described above. We incubated the allergen microarrays with
patient sera, and demonstrated that our method can successfully
distinguish among serum samples. Figure 3 shows that some
individuals have specific IgE to almost all allergens (e,f), while
others to only a few (d) or none at all (c). Diluting two serum
samples over a range of 50% to 3%, showed a predictable and
consistent decrease in signal intensity, with similar trends across
different allergen spots (Fig. 4).
We assessed the reproducibility of the microarray, by performing replicate tests using sera from six individuals with a wide range
of allergen-specific IgE levels. Analysis of variance (ANOVA)
showed that the identity of the allergen and source of the serum
were responsible for most of the observed variation in the data
(16.71% and 50.09%, respectively), and that array-to-array and
slide-to-slide effects accounted for only 0.12% and 0.85%, of the
variation respectively (Table 2). Regression analysis indicated that
pair-wise replicates were highly correlated (median R2 = 0.93,
Fig. 5a) and across all replicate pairs, the median of the slope of
the regression (beta) was 0.93 (Fig. 5b). Interestingly, 80.33% of
outlying values for beta, represented individuals who had overall
very low allergen-specific IgE indicating that concordance was
even higher in samples where true signals overpower noise.
Median coefficient of variances (CV) measured 0.09 to 0.15 for
comparisons of six arrays on the same slide, 0.11–0.20 for
comparisons between slides, and 0.14–0.25 for comparisons
between assays performed on three consecutive days (Table 3),
all within the variance reported previously for recombinant
allergen arrays [4,16]. Measurements of CV were not statistically
Table 3. CV of replicates.

Intra-slide

Inter-slide

Inter-assay

Surface

0.15

0.20

0.25

Cytoplasm

0.11

0.11

0.14

Pollen extract


0.11

0.17

0.10

0.15

0.19

Recombinant

Figure 5. Reproducibility of the allergy microarray. Six individuals were serially tested 18 times for 80 allergens on the allergen
microarray. A) Representative correlation plot of corrected fluorescence
intensity values (circles) of one serum sample binding to 80 allergens on
two replicate arrays. B) Histogram of slopes of regression curves
calculated for 918 pair-wise comparisons of replicates.

0.09

Non-pollen extract

0.14

0.16

0.23

Median coefficient of variation (CV) of fluorescence intensity reported for pollen
surface fractions (n = 21), pollen cytoplasmic fractions (n = 21), pollen
commercial extracts (n = 21), non-pollen commercial extracts (n = 9), and
recombinant allergens (n = 5). Only values where the mean of the replicates was
.0.5 were analyzed.

6



Figure 6. Allergen microarray testing vs clinical diagnosis of patients. Heatmap depiction of microarray results of six patients who were
diagnosed as either not having pollen allergy, but having some other aeroallergy (P2/O+) or as having both pollen and other aeroallergy (P+/O+).
Diagnoses were made at the in vitro allergy lab by ImmunoCAP RAST on 12 of the same (*) allergen species as on the array, four similar (+) allergen
species as on the array, and five other weeds not present of the array. All other allergens on the allergen array were not tested by ImmunoCAP RAST.

different for our cytoplasmic pollen extracts, commercial extracts,
or recombinant major allergens. Pollen surface fractions, however,
showed significantly more variability (p,0.05), perhaps reflecting
protein aggregation or instability. Nonetheless, the reproducibility
we observed across arrays, coupled with the strong correlation
between signals and serum concentration, indicate that the
allergen microarray can measure allergen-specific IgE over a wide
range of concentrations.
To evaluate the clinical validity of our method, we tested sera
from six patients who were diagnosed at the University of Chicago
in vitro allergy lab as having either i) no pollen allergy, but some
other aeroallergy or ii) both pollen and other aeroallergy. These
diagnoses were based on ImmunoCAP RAST (see Methods). We
observed that the allergen microarray results reflect the clinical
diagnoses of patients, detecting pollen allergen-specific IgE only in
patients who were diagnosed as pollen allergic (Fig. 6).
To further test the clinical validity of our method, we compared
results obtained by the allergy microarray with those obtained by
ELISA, an assay commonly used for clinical diagnosis. We
measured specific IgE in 40 human sera, observing binding to
cytoplasmic protein fractions and commercial extracts from six
allergens: grasses (Bermuda grass and Timothy grass), weeds
(mugwort and ragweed), and trees (birch and cottonwood) (see
Methods). The allergen microarray detected a positive signal for
81% (26/32) and 97% (29/30) of the signals detected by ELISA
for the cytoplasmic fraction and commercial extracts, respectively.

However, for about 50% of cases, IgE was detected with the
allergen microarray, but not with ELISA (Table 4). While it is
possible that these signals are false positives, our conservative
approach for setting thresholds (which controls the false positive
rate at 1%) supports the alternate possibility that the microarray is
more sensitive than ELISA. Further work using well-characterized
patients is needed to fully compare these assays.

Hierarchical clustering of allergen-specific IgE levels
We used the allergen microarray to investigate allergen-specific IgE
in two populations that are likely to be seen in an allergy clinic: 100
individuals with high levels of total IgE (.300 kU/L) and 76 allergic
individuals from the in vitro allergy lab at the University of Chicago.
To examine possible patterns in allergen-specific IgE, we
performed hierarchical clustering on allergen microarray-generated data. Analysis of specific IgE to five recombinant allergens,
which included a mite (Der p 1), a mold (Alt a 1), a grass pollen (Phl
p 2), a weed pollen (Amb a 1), and a tree pollen (Bet v 1), revealed a
striking clustering according to the phylogeny of the allergen
source. The three pollens clustered together, but differentiated
between monocots (Phl p 2) and dicots (Amb a 1 and Bet v 1)
(Fig. 7a). When this analysis was limited to only non-pollen
allergen extracts, including two molds and six animals including
two mites, two cockroaches, and two mammals (cat and dog), a
similar pattern was observed. Remarkably, not only did the pairs
of most closely related species cluster together, but the dendrogram reconstructed the phylogeny of these species, with the molds
clustering away from the animals, and within the animals, the
arthropods (mites and cockroaches) clustering away from the
mammals (Fig. 7b). Although clustering was less pronounced
when the analysis was limited to only pollen allergens, five of the
six grasses, which are monocots clustered together away from trees
and weeds, which are dicots (Fig. 7c). Although these results could
be explained by cross-reactivity [35,36], the fact that the allergen
microarray-generated data followed these patterns is quite
surprising. Previous studies examining cross-reactivity among
allergens have studied similarity of sequence and protein structure
among individual proteins, while we test for specific IgE to a
mixture of potentially allergenic proteins. Additionally, studies
have indicated that phylogenetically distantly related species such
as mite and shrimp can be cross-reactive due similar allergenic
proteins [37]. Although it is possible that there is one major
allergen that is driving the clustering of allergen-specific IgE data,
it is more likely that a combination of several related proteins
contributes to the clustering that we observed.

Table 4. Concordance of IgE detection by allergen microarray
and ELISA.

Cytoplasmic fraction
ELISA+

ELISA2

Total

Commercial extract
ELISA+

ARRAY+

26 (0.11)

116 (0.48) 142 (0.59) 29 (0.12)

ARRAY2

6 (0.03)

92 (0.38)

Total

32 (0.14)

208 (0.86) 240 (1.0)

98 (0.41)

ELISA2

Total

135 (0.56) 164 (0.68)

1 (0.01)

75 (0.21)

30 (0.13)

210 (0.87) 240 (1.0)

76 (0.32)

Specific IgE was measured for six allergens in 40 human sera using ELISA, an
assay commonly used for clinical diagnosis. Cytoplasmic fractions and
commercial extracts of each allergen were assayed. We scored each serum as
positive (+) or negative (2) for specific IgE to each allergen by ELISA and
allergen microarray using empirically defined detection thresholds (Methods).
The results are cross-tabulated here, showing the counts of assays positive by
one technique, positive by both techniques, and negative by both techniques
(proportion of total assays in parentheses).


7



Figure 7. Clustering of allergen-specific IgE levels. Hierarchical clustering of allergen-specific IgE to A) recombinant, B) non-pollen, and C)
pollen allergens. Within the pollens, grasses are indicated in green, trees in black, weeds in blue, and cedar (a gymnosperm) in red.

contrast, no significant differences were noted for Bermuda grass
or olive, while for ryegrass, the amount of specific IgE to the
commercial extract was significantly higher than to our cytoplasmic fraction (p = 2.1161028). For other allergens that were not
tested on the dot blots, the amount of specific IgE to commercial
extracts tended to be higher than to cytoplasmic fractions, perhaps
reflecting greater purity or a higher proportion of allergenic
proteins to non-allergenic proteins in these particular batches of
commercial extracts (Fig. 8a).
While the amount of specific IgE to pollen surface was not as
high as to other fractions, a substantial number of individuals
(25%) had high amounts (.1.0 microarray units) to at least one
surface extract. The most allergenic surface allergens were elder,

Comparison of specific IgE to surface, cytoplasmic and
commercial extracts
Comparison of the relative amounts of specific IgE among
individuals to the different pollen extracts (surface, cytoplasm, and
commercial extract), revealed that, as a class, the amount of
specific IgE to cytoplasmic proteins was not significantly
different from the amount of specific IgE to commercial pollen
extracts (p.0.10). However, many individual allergens showed
differences, including those tested in the dot blot experiments
(Fig. 8a). Among these, individuals had significantly higher
amounts of specific IgE to the cytoplasmic fraction of
ragweed (p = 1.5961023), pecan (p = 4.7161024), and cedar
(p = 1.10610211) as compared to the commercial extracts. In

8



Figure 8. Comparison of specific IgE to surface, cytoplasmic and commercial extracts. Bar graphs comparing levels of specific IgE in
microarray units to surface fractions (black), cytoplasmic fractions (grey), and commercial extracts (white) of different pollen allergens. A) Bars
represent the mean of 176 individuals with standard error bars. Cases where specific IgE to commercial extracts was significantly greater than to
cytoplasmic extracts are denoted by a black asterisk (*) and cases where specific IgE to cytoplasmic fractions was significantly greater than to
commercial extracts is denoted by a plus sign (+). B) Bars represent the mean of 18 replicates (of individual 85806) with standard error bars.

used or to less efficient extraction of proteins from these species.
Indeed, due to the highly lipophilic nature of pollen surface
proteins, effective isolation remains challenging.

ragweed, ash, bluegrass, and elm (Fig. 8a). In fact, some serum
samples showed extensive sensitization to surface fractions, with
cases of higher amounts of specific IgE to surface fractions as
compared to commercial extracts (Fig. 8b). These data are
consistent with observations made on the gels and dot blots,
indicating that the pollen surface, which has been largely
overlooked, contains allergens. Lack of detectable specific IgE to
certain surface fractions could be due to the serum population

Comparison of specific IgE to recombinant allergens and
total protein extracts
To directly test the utility of screening recombinant allergens,
rather than extracts containing a mixture of proteins, we
9



included five recombinant major allergens on the allergen array;
Bet v 1 from birch, Amb a 1 from ragweed, Phl p 2 from Timothy
grass, Der p 1 from mite, and Alt a 1 from mold. While the
recombinant major allergen from mite, ragweed, and birch
captured over 80% of sensitized individuals, for all species, there
were several individuals who had specific IgE to commercial
extracts, but not to the corresponding recombinant allergen.
The least informative recombinant allergens were from mold
(Alta a 1) and Timothy grass (Phl p 2) for which 73% and 70% of
allergic individuals had specific IgE to commercial extracts but
not the recombinant major allergen, respectively (Fig. 9). These
results imply the presence of another allergen in these species to
which the majority of people have specific IgE. Alternatively,
post-translational modifications present in plant cell extracts
may be missing in the recombinant proteins, especially if they
were expressed in a non-eukaryotic system. Conversely, 27–45%
of individuals had specific IgE to the major recombinant
allergen from birch (Bet v 1), ragweed (Amb a 1), and mite (Der
p 1), but not to the total protein extracts. These results suggest
that the relative concentration of the major allergen is
substantially lower in the total protein extract than the
recombinant allergen.

providing an efficient and economically feasible assay to measure
allergen-specific IgE in sera.
Previous studies explored this possibility by focusing on a few
recombinant allergens and have suggested that microarrays
containing recombinant allergens offer advantages over those
containing native protein extracts [4,7,9–11,38]. They argue that
1) easier standardization of recombinant allergens results in better
reproducibility across assays and 2) that recombinant allergens
offer very specific diagnosis, identifying disease-eliciting molecules
that can be used for more effective immunotherapy. However, we
showed that our microarray is highly reproducible whether
allergens are recombinant or present in native extracts.
Additionally, limiting analysis to recombinant major allergens
potentially restricts diagnosis to just over half of the affected
population [39] and due to the lack of post-translational
modifications, some recombinant allergens may not display the
same immunological reactivity as their native counterparts [40].
Indeed, our results indicate that the recombinant major allergens
from Timothy grass (Phl p 2) and mold (Alt a 1) are not sufficient
to diagnose individuals for allergen sensitization. We also
successfully showed that pollen surface fractions, which have
largely been overlooked in the past, contain allergens. It is thus
important to pursue the identification, cloning, and characterization of these pollen surface allergens, and this is the subject of
another manuscript (in preparation). Nonetheless, the utility of
recombinant allergens remains clear, especially with regard to
molecule-specific diagnosis. Microarrays containing combinations
of extracts and recombinant allergens will likely be the most
beneficial diagnostic approach, combining comprehensive
testing and protein-specific analysis, as suggested by Fall and
colleagues [8].
Further refinement of the allergen microarray will likely
improve its utility for clinical and research applications. For
example, we observed significant autofluorescence that varied
from spot-to-spot and allergen-to-allergen, possibly caused by
differential spotting of fluorescent proteins, aggregated proteins, or
non-proteinaceous fluorescent contaminants in the extracts. While
a computational approach effectively corrected for autofluorescent
signals, some variation was undoubtedly introduced by this
phenomenon. Improved purification, solubilization, and quantification of proteins spotted on the microarray would likely make this
a more reliable assay. The use of a two-color antibody labeling
system similar to that described by Kattah and colleagues [41],
could also reduce variability. Finally, although we have shown that
our method reflects clinical diagnosis of patients (as determined by
ImmunoCAP RAST) and displayed high concordance with
ELISA in detecting the presence of allergen-specific IgE, it is still
important to establish the extent of the allergen microarray’s
reliability and clinical relevance by validating it using large
populations of well-characterized allergy patients. Ultimately, the
use of the allergen microarray in conjunction with assessment of
symptoms by a doctor, could greatly improve the accuracy and
efficiency of allergy diagnostics.
In addition to its application in the clinic, the allergen
microarray can be used as a research tool to quickly collect
phenotype data for genetic mapping of susceptibility genes.
Genetic studies have implicated several genes in allergy disposition, but most have not been consistently replicated across
populations due to the complex nature of the disease [42–44].
Measuring allergen-specific IgE with the allergen microarray may
facilitate quantitative trait analysis of allergy predisposition in
populations, particularly if the number of genetic and environmental factors influencing this intermediate phenotype is smaller
than the number of factors affecting the complex disease [45,46].

Discussion
Here, we developed a protein microarray containing 80
different allergen fractions from 31 different species of allergen
sources, and have demonstrated that it can reproducibly measure
allergen-specific IgE in small amounts of sera. With the continuing
advancement of protein microarray technology, this microarray
could be expanded to hundreds or even thousands of allergens,

Figure 9. Comparison of specific IgE to recombinant allergens
and total protein extracts. Bar graph comparing reactivity to
commercial extract vs. the corresponding recombinant major allergens
in microarray units. Bars represent the percent of individuals (mold:
n = 153, Tim: n = 143, Bir: n = 106, Rag: n = 124, Derp: n = 171) who show
positive reactivity to commercial extract but no reactivity to recombinant major allergen (black), positive reactivity to recombinant major
allergen but no reactivity to commercial extract (white), and positive
reactivity to both (grey).


10



Indeed, asthma linkage and association studies have benefited
from a similar approach [47].

data; and Matthew Cummings and Daniel Rosenbaum for technical
assistance.

Acknowledgments

Author Contributions

We thank Carole Ober, Nancy Cox, Anne Sperling, Dan Nicolae, Greg
Copenhaver, and George Arida for helpful comments and discussions;
Jacquelynne Corey and Khalil Ouyoun for providing sera from allergic
patients; Josef Jurek for developing the database housing all microarray

Conceived and designed the experiments: KAVC DP. Performed the
experiments: KAVC. Analyzed the data: KAVC DFC. Wrote the paper:
KAVC DFC DP. Wrote programs for data analysis: DFC. Supervised the
entire study: DP.

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