American Journal of Plant Sciences, 2011, 2, 476-483
doi:10.4236/ajps.2011.23056 Published Online September 2011 (http://www.SciRP.org/journal/ajps)
Purified Wheat Gliadin Proteins as Immunoglobulin
E Binding Factors in Wheat Mediated Allergies
Jacek Waga1, Krystyna Obtułowicz2, Jerzy Zientarski1, Ewa Czarnobilska2, Andrzej Skoczowski3
1Cereals Department, Plant Breeding and Acclimatization Institute, Krakow, Poland; 2Department of Clinical and Environmental
Allergology, Collegium Medicum Jagiellonian University, Krakow, Poland; 3Institute of Biology, Pedagogical University, Krakow,
Poland.
Received June 6th, 2011; revised August 15th, 2011; accepted August 22nd, 2011.
ABSTRACT
Some wheat gliadin proteins are strong allergens that may cause various symptoms of food allergies and baker’s
asthma. The most immunoreactive ω-5 gliadin fractions are the main allergens in wheat dependent exercise induced
anaphylaxis (WDEIA). While the allergenicity of ω-5 is quite well understood, knowledge about α, β, γ and ω-1.2 gli-
adins is much more scanty. This study examines allergenic properties of other fractions as compared to ω-5. Gliadins
were extracted from flour of winter wheat (Triticum aestivum L.) cultivar Ostka strzelecka. Purified samples represent-
ing proteins belonging to α, β, γ, ω-1.2 and ω-5 classes were isolated using preparative gel electrophoresis. Immuno-
reactivity and allergenic properties of these proteins were analyzed by ELISA using sera from allergic patients with
elevated sIgE (>2KU/L), and by skin prick test (SPT). ELISA showed that ω-5 and ω-1.2 differed considerabely from α-,
β- and γ-gliadins in respect of immunoreactivity. Responses of both ω-gliadins were almost twice as high as for other
fractions. Significant differences were also observed among individual ω-gliadin fractions as evidenced by ANOVA.
SPT showed that patient with symptoms of baker’s asthma and WDEIA had a positive results to all gliadins tested. An-
other patient with baker’s asthma (but not WDEIA) reacted positively only to ω-5 gliadins. In two patients with skin
allergy SPT were negative with all analyzed proteins. Results show ω-1.2 gliadins to be almost as immunorective as
ω-5. The α-, β- and γ-gliadins also recognize specific IgE antibodies, but their binding capacity is only about half that
of ω-fractions. This kind of immunoreactivity could still be important since a cumulative effect of individual fractions
may intensify disease symptoms in allergic patients.
Keywords: Allergies, Gliadins, Immunoreactivity, Preparative A-PAGE, Wheat
1. Introduction
Gliadin proteins are functional components of most wheat-
food products. However, for people with celiac disease
or specific wheat food allergies, they may be toxic or
allergenic factors. Since gliadins (as gluten proteins) are
important in human nutrition throughout the world, their
celiac toxicity and allergenicity are serious problems for
both medical and nutrition sciences.
Gliadins are unique, highly atypical and the most com-
plex and heterogeneous protein group in the plant kingdom.
They are composed of hundreds of monomeric proteins,
rich (like all prolamines) in proline (ca. 14%) and gluta-
mine (ca. 40%) [1,2]. The great polymorphism of gliadin
proteins is determined genetically. Gliadins are coded by
six polygenic loci on chromosomes 1 and 6 of the A, B,
and D genomes of hexaploid wheat (Triticum aestivum L.)
[3]. Each locus contain from several to tens of individual,
closely linked genes. Many individual gliadin genes have
been duplicated throughout evolution. Their structures
subsequently changed by point mutation, unequal cross-
ing-over, gene silencing, translocation, and deletion of
DNA fragments. Consequently, multiple alleles for each
locus arose during evolution of hexaploid wheat. Each
allele codes for specific groups of polypeptides defined
as gliadin protein blocks [4]. Genetic studies show pro-
teins of each block to be expressed and inherited together.
Thus, individual wheat genotype contains tens of distinct
monomeric gliadins, differing in sequence, chemical
properties, and physicochemical structure. Moreover,
gliadin polypeptides compose a number of combinations
among different wheat genotypes. Hence wheat species,
cultivars and breeding lines vary greatly in gliadin com-
position. This heterogeneity, as well as inter-varietal dif-
ferences, have been shown by acid polyacrylamide gel

Purified Wheat Gliadin Proteins as Immunoglobulin E Binding Factors in Wheat Mediated Allergies477
electrophoresis (A-PAGE), high performance capillary
electrophoresis (HPCE) and reversed phase high per-
formance liquid chromatography (RP-HPLC) [5-7].
Gliadins also have unique biochemical structures,
containing both repeating and non-repeating sequences.
For example, α-gliadins are rich in short repeated se-
quences localized near the N-terminal regions, while
their C-terminal regions are unique, rich in glutamine and
contain six cysteine residues that form intra-molecular
disulphide bonds [8-10]. In contrast, ω-gliadins have no
cysteine residues and consist mainly of short, repeated
sequences, with the tetrapeptides QPQQ and PQQP be-
ing most frequent (23 and 30 repetitions respectively)
[11]. Repeated sequences often form epitopes that bind
IgE antibodies present in elevated amounts in sera of
patients sensitive to gluten. Multiple epitopes may am-
plify formation of IgE-gliadin complexes and―in con-
sequence―protein immunoreactive properties.
It seems obvious that such great differentiation can
significantly influence allergenicity of individual proteins
within the protein complex. The allergenic properties of
ω-5 gliadins are rather precisely known. These are the
main allergens in wheat-dependent exercise induced
anaphylaxis (WDEIA), the gluten protein allergy most
dangerous for human life [12,13]. However, there have
been far fewer studies of α, β, γ and ω-1.2 gliadins. These
proteins may also significantly impact allergenicity, since
all gliadins share many related sequence characteristics
and epitopes. Thus, the allergenicity of the entire protein
complex may result from additive effects of its individual
components. In addition, each of five α, β, γ, ω-5 and
ω-1.2 gliadin classes is composed of several proteins,
appearing as individual bands upon A-PAGE. No de-
tailed studies have been performed to identify the most
allergenic fractions among α, β, γ, ω-5 and ω-1.2 groups.
It seems probable that individual gliadin fractions may
also differ from each other in allergenicity; this may be
important in allergology to identify major allergens im-
munologically active in various allergies, and in plant
breeding for development of wheat genotypes having
decreased allergenic properties.
Recent studies have considerably advanced the under-
standing of cereal proteins immunology, but knowledge
of wheat gliadins as allergens is still incomplete [14].
Important aspects concerning protein structure to aller-
genicity remain unexplained. One such aspect is the rela-
tion of allergenic properties to polymorphism. Studies on
allergenicity of gluten proteins are multidisciplinary,
with medical and biochemical sciences playing major
roles in this field. Medical sciences are usually directed
to characterize symptoms of allergic diseases among
large populations of subjects sensitized to predicted al-
lergens. Biochemical research has attempted to identify
the most immunoreactive peptides and epitopes among
allergenic proteins. The aim of the present study―more
biochemical than medical in approach―is to isolate and
characterize immunoreactive properties of individual,
purified gliadin proteins, belonging to α, β, γ, ω-5 and
ω-1.2 groups. Special attention was paid to the aller-
genicity of α, β, γ, and ω-1.2 as compared to ω-5 gliadin
fractions.
2. Material and Methods
2.1. Plant Material
The winter wheat cultivar Ostka strzelecka, from the
collection of Plant Breeding and Acclimatization Insti-
tute, Cereals Department, Krakow (PBAI), harvested in
2008, was used in this study. Kernels were milled on a
Brabender Quadrumat Senior mill (Brabender GmbH,
Germany) having sieves with mesh diameters from 150
μm to 500 μm. The resulting flour was used for gliadin
extraction.
2.2. Purification of Gliadins by Preparative
A-PAGE
Gliadins were extracted from flour with 70% EtOH (1:10
v/w) and gentle shaking on a Grant-bio PTR-60 rotator at
15 rpm/min overnight at room temperature. After cen-
trifugation (12,500 x·g, 10 min), extracts were introduced
into aluminium lactate buffer (pH = 3.1) saturated with
sucrose in the proportion 1:1. An aliquot (2.7 mL) of this
was applied to the surface of a polyacrylamide gel col-
umn (T = 8%, C = 0.29%) in a Model 491 PrepCell elec-
trophoretic apparatus (BioRad, USA) and gliadins were
fractionated by A-PAGE [15]. The total amount of gli-
adin proteins in extracts applied to the column was ap-
proximately 10 mg. The gel column was 10 cm high and
37 mm wide. Separations were done at constant power
(11 W); maximum voltage was set at 320 V, and current
varied during electrophoresis from 41 to 19 mA. At the
bottom of the gel column an elution chamber equipped
with frits (BioRad patent 4.877.510) was installed. The
elution chamber was connected via a silica connector
with a chamber containing eluent solution (0.01 M acetic
acid). Fractions leaving the column were collected be-
ginning two hours after the start of electrophoresis.
Separated proteins were transferred from the elution
chamber to a fraction collector (Model 2110, BioRad,
USA) with a peristaltic pump. It was thus possible to
collect 80 aliquots from a protein mixture during 12
hours.
Aliquots from preparative separations were tested
twice by analytical A-PAGE [16]. The first separation, to
identify gliadin fractions, was done in a Desaphor Elec-
trophoretic Chamber (Desaga GmbH, Germany). 200 μL

Purified Wheat Gliadin Proteins as Immunoglobulin E Binding Factors in Wheat Mediated Allergies
478
of each aliquot was mixed with 90 μL of aluminium lac-
tate buffer (pH = 3.1) saturated with sucrose, and intro-
duced into electrophoretic wells in the polyacrylamide
gel. Proteins were separated for four hours at constant
voltage (500 V). Based on the obtained results, aliquots
containing identical protein bands were combined and
concentrated to 1.5 mL with a CentriVap Concentrator
(Labconco, USA). Resulting fractions were then again
electrophoresed by A-PAGE using Mini Protean 3 (Bio-
Rad, USA) to check their purity. The second separation
was done for 130 minutes at constant voltage (155 V);
current varied during electrophoresis from 12 to 9 mA.
The protein content of each fraction was determined by
the Bradford method using Bio-Rad Protein Assay Kit I
(Bio-Rad, USA). Purified protein samples for further
analysis were then lyophilized and stored at 4˚C. Using
these methods, it was possible to obtain 0.3 mg to 1.0 mg
of individual fractions.
2.3. Patients
Four patients (two men and two women, ages 25 - 55)
with increased sIgE serum level to gluten (> 2KU/L,
UniCap Pharmacia, Sweden), treated in Outpatients De-
partment of Allergology in University Hospital in Kra-
kow, were included in this study (Table 2). Two of these
subjects were bakers with baker’s asthma, developed at
work from exposure to wheat dust. One of these also
developed anaphylaxis after consuming whole grain
bread (gluten ingestion) after physical exercise [WDEIA].
The serum level of sIgE to gluten in both patients was
high (11.4 KU/L and 12.3 KU/L, compared to the normal
value of < 0.35 KU/L). The third patient, exhibited atopic
dermatitis and atopic eczema, was more severe after skin
contact with wheat flour. Patient four also exhibited skin
symptoms (urticaria) after skin contact with wheat flour.
The serum level of sIgE to gluten in third and fourth pa-
tients was elevated but much lower than in patients with
bronchial asthma (2.12 KU/L and 3.36 KU/L).
2.4. ELISA
Binding capacity of purified gliadin fractions by IgE an-
tigliadin antibodies from allergic patients sera was ana-
lyzed by immunoenzymatic indirect ELISA. Proteins
were diluted in carbonate buffer (0.05 mol/L, pH = 9.6)
and microtiter wells were coated with 100 μL of ana-
lyzed solutions overnight at 4˚C. Protein concentration of
all samples was adjusted to 100 μg/mL. Each sample was
analyzed in four replicates. In blank samples 1% foetal
calf serum (FCS) diluted in phosphate-buffered saline
with addition of 1% Tween (PBST), was introduced into
the wells instead of gliadin samples. On the next day all
wells of the microtiter plates were blocked with 1% FCS
at room temperature for two hours. After removing the
blocker, pooled sera of all patients, diluted 1:5 in PBST,
were introduced to microtiter wells. Plates were incu-
bated for two hours at 37˚C. After incubation the sera
were removed and 100 μL of the secondary antibody
solution (ξ-chain of IgE antihuman antibodies conjugated
with horseradish peroxidase, Sigma Co., USA) diluted
1:1000 in PBST was added to each well and incubated
again for two hours at 37˚C. After each stage of ELISA,
wells were rinsed three times with PBST. The substrate
for horseradish peroxidase was O-Phenylenediamine
prepared according to manufacturer’s instruction (Sigma
Co., USA). Reaction with substrate was carried out for
20 minutes in darkness, and stopped by adding 100 μL of
3M H2SO4 to each well. Optical density (OD) of col-
oured products of the immunoenzymatic reaction was
estimated with an ELISA plate reader (Opsys MR,
Thermolab System, USA) at 490 nm. Average values
from four repetitions, multiplied by 1000, were accepted
as indicators of protein immunoreactivity. Statistical sig-
nificance of differences among average values of the
ODx1000 indicators was estimated by one dimensional
ANOVA. Least significant difference (LSD) intervals at
P = 95% were estimated by the Tukey test. ANOVA and
Tukey tests were performed using the program Stati-
graphics Plus 1.4.
2.5. Skin Prick Tests
In all patients studied, skin prick tests (SPT) were per-
formed according to standard method [17] with prepared
gliadin fractions in concentration 200 ug/ml, as well as
with positive (1% histamine) and negative (Allergopharma,
Germany) control. The skin reactions were evaluated
after 15 minutes: wheals ≥ 3 mm diameter were judged
positive at negative control―0 mm and positive con-
trol―3 mm or more.
3. Results
A-PAGE of gliadin proteins from winter wheat cultivar
Ostka strzelecka reveals 25 bands (Figure 1).
The ω-5 and ω-1.2 groups contain three and five bands
respectively. All ω-gliadins are composed on single
bands, well-marked and clearly separated from each
other. The remaining groups of α-, β- and γ-gliadins con-
tain seven, six and four bands respectively. Some bands
are much thicker and closer than in ω fractions. That is
because the α, β and γ zones of A-PAGE electrophore-
grams commonly superimpose many polypeptides having
similar or identical mobilities, making identification and
purification a serious methodological problem.
Preparative A-PAGE fractionated gliadins from Ostka
Strzelecka into 80 aliquots, which were than analyzed by
analytical A-PAGE. Aliquots with identical protein

Purified Wheat Gliadin Proteins as Immunoglobulin E Binding Factors in Wheat Mediated Allergies479
compositions were combined, concentrated and lyophi-
lized. Twelve purified protein samples were thus ob-
tained for further studies (Figure 2).
Figure 1. A-PAGE electrophoregrams of gliadin patterns of
winter wheat cultivar Ostka strzelecka. α, β, γ, ω-5 and ω-1.2
(indicated on the left hand side of the electrophoregram):
groups of gliadin proteins varied in regard of electropho-
retic mobility.
Figure 2. A-PAGE electrophoregram of purified gliadin
fractions obtained from winter wheat cultivar Ostka strze-
lecka. OS-pattern of gliadin proteins complex specific for
cultivar Ostka strzelecka. 1, 2, 3 ··· 12 (on the top of the pic-
ture): the number of the runs. α[1], β[1], β[2] ··· ω-1.2[3]
(under the protein bands): symbols of individual gliadin
protein fractions.
One of these samples contained α-gliadins, three con-
tained β-, two γ-, three ω-5 and three contained ω-1.2 gli-
adin fractions. The first sample (α[1]) was composed on
two α-gliadin bands. The next 3 samples comprised
β-gliadins (2-4: β[1], β[2] and β[3]). Two of these (β[1]
and β[3]) were composed on single bands whereas the
third (β[2]) combined two bands: β[1] and β[3]. Samples 5
and 6 contained γ-gliadins. Sample 5 (γ[1]) has an intense
band with several superimposed proteins―products of
genes localized on different chromosomes, plus some of
the β[3] band; sample 6 appears to contain only one
γ-gliadin (γ[2]). Two subclasses of the ω-gliadins (ω-1.2
and ω-5) were examined separately. For each subclass
three samples (7, 8, 9 and 10, 11, 12) were obtained; each
sample contained one major band (ω-5[1], ω-5[2], ω-5[3]
and ω-1.2[1], ω-1.2[2], ω-1.2[3]).
Indirect ELISA showed large differences among im-
munoreactive properties of twelve purified gliadin frac-
tions, as revealed by binding capacity to serum IgE anti-
bodies of patients allergic to gluten. ANOVA confirmed
these differences to be statistically significant (P < 0.001)
(Table 1).
For all average OD values, least significant difference
(LSD, P = 95%) and high and low limits of average val-
ues were calculated by the Tukey test. Observed OD
values for subclasses ω-1.2 and ω-5 were almost twice as
high as for other protein fractions. The highest OD val-
ues were for fractions ω-5[2] and ω-1.2[3], and the low-
est for γ[1] (Figure 3).
OD variability ranges of most α-, β- and γ-proteins did
not differ significantly however, γ(1) was significantly
lower in OD value than α(1), β(1) and β(3) fractions.
Many significant differences occurred among ω-gliadin
fractions. The average OD value for ω-1.2[1] was signifi-
cantly lower than for ω-5[1], ω-5[2], ω-1.2[2] and
ω-1.2[3]. Similarly, the value for ω-5[3] was significantly
lower than for ω-5[1], ω-5[2] and ω-1.2[3], while the
value for ω-1.2[2] was significantly lower than for ω-5[2]
and ω-1.2[3].
Table 1. One way analysis of variance for average OD val-
ues obtained by ELISA of purified gliadin fractions from
winter wheat Ostka strzelecka using pooled sera of four
patients allergic to gluten.
Source of variationSS df Variance F
Overall 515218 35
Between groups
(proteins) 496219 11 45111 56,96***
Among groups
(error) 18999 24 792
SS―sum of squares; df―degrees of freedom; ***―significant at P < 0.001.

Purified Wheat Gliadin Proteins as Immunoglobulin E Binding Factors in Wheat Mediated Allergies
480
Allergenic properties of five chosen purified gliadins
(α[1], β[1], γ[2], ω-5[2] and ω-1.2[3]), were evaluated by
skin tests (SPT) on patients who also donated serum for
ELISA. Patient 1, with symptoms of baker’s asthma and
WDEIA and the highest specific IgE level, had positive
results for all fractions tested (Table 2).
Patient 2, having symptoms of baker’s asthma but not
WDEIA, and having a specific IgE level similar to pa-
tient 1, reacted positively only to gliadin ω-5[2]; this
fraction was also most immunoreactive by ELISA. Pa-
tients 3 and 4 (with skin symptoms and low levels of
specific IgE antibodies) showed SPT wheals slightly lar-
ger than the negative control, but this could not be evalu-
ated as a positive reaction.
Figure 3. Average optical densities (OD values) obtained by ELISA, and LSD intervals (P = 95%) estimated by Tukey test for
purified gliadin fractions of cultivar Ostka strzelecka in reaction with human sera from patients allergic to gluten. α[1], β[1],
β[3],···,ω-1.2[3] (on the X-axis): Symbols of purified gliadin fractions (compatible with Figure 2). 250, 350,···,650 (on the
Y-axis): absorbance (OD) values of ELISA multiplied by 1000 used as gliadin’s immunoreactivity indicators (the higher OD
value the highest immunoreactivity of the gliadin fraction).
Table 2. Results of skin prick tests performed for patients allergic to gluten (sIgE-f76 > 2.0 IU/mL) using individual gliadin
fractions as antigens.
Wheal diameter (mm)
Patients sIgE (IU/L) Symptoms of disease
α β γ ω5 ω1.2 NC PC
1 12.3 Baker asthma, WDEIA >3 >3 >3 >3 >3 0 >3
2 11.4 Baker asthma 2 2 2 >2 2 0 >3
3 3.36 Skin symptoms, AD 1.5 1.5 1.5 1.5 1.5 0 >3
4 2.12 Skin symptoms, URT 1.5 1.5 1.5 1.5 1.5 0 >3
NC―Negative control (0.9% NaCl solution); PC―positive control (1% histamine); AD―atopic dermatitis; URT―urticaria.

Purified Wheat Gliadin Proteins as Immunoglobulin E Binding Factors in Wheat Mediated Allergies481
4. Discussion
In the present studies we aimed to obtain information
about immunoreactive properties of isolated and purified
gliadin fractions. Therefore we observed the effects of
immunological reaction between individual proteins and
IgE antibodies using pooled sera of four patients showing
different symptoms of gluten protein intolerance for
ELISA. Immunoreactive properties of characterized gli-
adins were then verified using skin prick test on patients
who also donated sera for this study. In spite of the fact
that patients 1 and 2 (in accord with Table 2) showed
significantly higher sIgE levels than patients 3 and 4,
each having antibodies which increased the total amount
of sIgE in the polled sera. However, antibodies present in
sera of patients showing various symptoms of wheat glu-
ten protein allergies may slightly differ each other in re-
spect of amino acid sequences in hypervariable regions
and―in consequence―may bind to different epitopes of
gliadin polypeptides. Hence, we expected pooled sera to
show broad immunological activity, and antibodies of
each patient contribute to immunoreactivity of analyzed
proteins as measured by ELISA. A similar strategy has
been used in other studies on wheat protein allergenicity.
Watanabe et al. [18] identified allergenic peptides in glu-
ten using serum of one patient, while Tanabe et al. [19]
used pooled sera of four patients to show that peptide
QQQPP, common in gluten proteins, is a strong IgE
binding epitope.
The complexity of gliadin proteins complicates efforts
to recognize and characterize the biochemical bases of
gliadin allergenicity. We are convinced that studies of
individual, native gliadin fractions can provide more ob-
jective and valuable information about their immunore-
activity than can analysis of the whole gliadin complex
or even separate α, β, γ and ω classes. We therefore in-
vestigated allergenic properties of highly purified pro-
teins isolated by preparative A-PAGE. This method is
useful for analysis of gliadin’s immunoreactive proper-
ties, as confirmed in our previous studies and by others
[15,20]. It is thus possible to obtain purified fractions that
have the immunological characteristics for native pro-
teins; preparative A-PAGE permits isolation of proteins
in amounts sufficient for immunoassays (ELISA and
SPT), as described in this paper.
Results in Figure 3 reveal relationships of both gliadin
classes (α, β, γ, ω-5 and ω-1.2) and individual gliadin
fractions to immunoreactivity. Observed differences in
immunoreactive properties among gliadin classes permit
dividing the analyzed proteins into two groups: the
ω-proteins (ω-5 and ω-1.2) and the remaining α, β and γ
proteins. Immunoreactivity of the ω-gliadins was much
higher than that of the other gliadins. Our ELISA results
confirm the highest IgE binding capacity for the class
ω-5. These results were analogous to those of others
[12,13,21]. We found, however, that ω-1.2 gliadins have
immunoreactivity similar to or even greater than ω-5.
The α, β and γ gliadins may also bind specific IgE anti-
bodies of patients allergic to gluten. Their immunological
activity is much lower than that of both ω subclasses, but
could be important especially, if there is an additive ef-
fect of individual proteins on disease development. This
assumption is partly strengthened by the results of pro-
teomic studies of Akagawa et al. [22] who showed that
γ-gliadins may be important wheat flour allergens using
pooled sera from seven patients allergic to gluten.
We must emphasize that our results show previously
undescribed differences in immunoreactivity among in-
dividual α, β, γ, ω-5 and ω-1.2 gliadins. This relationship
is especially evident among ω-5 and ω-1.2, where ω-5[2]
was much more immunoreactive than ω-5[3]. Differ-
ences between ω-1.2[1] and ω-1.2[3] fractions were even
greater. Moreover, one ω-1.2 fraction (ω-1.2[3], a weak
protein band) was more immunoreactive than ω-5[3].
Both ω gliadin subgroups, like other gliadins, are highly
polymorphic protein groups [23,24], coded by genes on
different chromosomes (ω-1.2―mainly chromosome 1D,
and ω-5 on chromosomes 1A and 1B). The catalogue of
gliadin proteins worked out by Metakovsky [25] shows
46 ω-gliadin blocks, each composed on several fractions
of different physicochemical properties. Blocks coded by
different chromosomes may occur in all possible combi-
nations among wheat genotypes. This emphasizes the
complex structures and major differences among ω-gli-
adins. The great diversity, as in other gliadin classes,
may relate to different immunoreactive properties, and
may make possible to identify the main wheat allergens
responsible for pathogenesis in allergic diseases. This
could also permit breeding strategies for development of
hipoalergenic wheat genotypes, since knowledge about
allergenicity differentiation of individual gliadin frac-
tions may greatly facilitate selection of hybrid plants
containing proteins of decreased immunoreactivity It has
been proposed that ω-5 gliadins (native or recombinant)
may serve as the basis of an effective test to identify and
diagnose food allergy in patients sensitive to gluten
[26-29]. Since ω-gliadins differ in immunoreactivity,
choosing proper fractions for assays may significantly
improve test sensitivity and specificity; isolation, purifi-
cation and analysis of purified gliadin allergenicity may
be important in optimizing such tests. Supposedly, use of
less allergenic α-, β- and γ-gliadins in screening may also
reveal significant information, indicating specific suscep-
tibility to minor wheat allergens.
Our results suggest that research on immunoreactive
properties cannot be restricted to gliadin groups (α, β, γ,

Purified Wheat Gliadin Proteins as Immunoglobulin E Binding Factors in Wheat Mediated Allergies
482
ω-5 and ω-1.2), but should be extended to individual
protein fractions (A-PAGE bands) within groups. Con-
sidering that many individual A-PAGE bands can be
further fractionated by two-dimensional (IEF/SDS-PAGE)
electrophoresis (results unpublished), one may suppose
that identification of the most allergenic proteins among
ω-gliadins (and other gliadin groups) should be carried
out on the level of individual monomeric proteins.
Results of our in vitro ELISA tests were confirmed to
some extent by SPT; both methods showed an immu-
nological response to applied protein fractions. Patient 1
with baker’s asthma and WDEIA displayed a positive SPT
reaction to all gliadins. However, in the patient 2 with
baker’s asthma but not WDEIA, only fraction (ω-5 [2])
was immunoreactive, in spite of levels of sIgE antibodies
being similar (12.3 kU/L and 11.4 kU/L respectively) in
both patients. In patients with skin symptoms, no positive
results were observed. These reactivities suggest that ω-5
gliadins, strong allergens in WDEIA, may be also immu-
nologically active in baker’s asthma. Previously, major
allergens in this disease were thought to be albumins that
inhibit trypsin and α-amylase [30,31]. These observations
also suggest specificity between various antigens and the
type of allergic disease that they may trigger. Considering
that our research was performed on only four patients,
verification of these observed relationships in population
studies with larger groups comprising more gluten protein
susceptible persons is necessary, and will be a goal of our
further research.
Based on the obtained results the following conclu-
sions can be drawn:
 The α, β, γ, ω-1.2 and ω-5 gliadin groups obtained
from winter wheat cultivar Ostka strzelecka show
significant differentiation in regard of immunoreac-
tive properties as measured by ELISA using pooled
sera of four patients with gluten intolerance.
 Immunoreactivity of both ω-1.2 and ω-5 gliadin sub-
groups is much higher than that of the other α-, β- and
γ-gliadin groups.
 Some of individual gliadin fractions among gliadin
groups and subgroups differ also significantly in re-
gard of immunoreactivity. One of the ω-1.2 gliadins
was even more immunoreactive than ω-5[3] fraction
what was undescribed previously.
 Purified gliadin protein fractions used as reagents for
skin prick tests show differentiated immunological
response in on patients who donated sera for the study,
hence they may serve as the as the basis of an effec-
tive test to identify and diagnose food allergies.
5. Acknowledgements
This work was carried out with financial support from
The Polish Ministry of Science by research grant No. N
N310 162238.
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