Introduction. The number of people with celiac disease is rapidly increasing. Gluten, is one of the most common food allergens, consists of two fractions: gliadins and glutenins. The research objective was to determine the optimal conditions for estimating gliadins by using enzyme-linked immunosorbent assay (ELISA). Study objects and methods. The experiment involved wheat flour samples (0.10, 0.20, 0.25, 0.50, and 1.0 g) suspended in different solvents (ethanol, methanol, 1-propanol, and isopropanol) of different concentrations (40, 50, 60, 70, 80, and 90% v/v). The samples were diluted with Tris buffer in ratios of 1:50, 1:100, 1:150, and 1:200. The gliadin test was performed using a Gliadin/Gluten Biotech commercial ELISA kit (Immunolab). Results and discussion. The optimal conditions for determining gliadin proteins that provided the highest gliadin concentration were: solvent – 70% v/v ethanol, extract:Tris buffer ratio – 1:50, and sample weight – 1.0 g. Conclusion. The obtained results can be of great importance to determine gliadin/gluten concentrations in food products by rapid analysis methods.
Extraction, gluten, gliadins, wheat flour, enzyme-linked immunosorbent assay (ELISA)
INTRODUCTION
Gluten is the one of the most common food allergens.
According to the Codex Alimentarius [1], gluten is
defined as a protein fraction of wheat, rye, barley, oats,
their cross varieties, and derivatives, which some people
are sensitive to [2]. Gliadins and glutenins are two
fractions present in approximately equal amounts in
gluten [3].
Gliadins are respresented by monomers. Due to the
high content of glutamine and proline, these proteins
are also called “prolamins” [4, 5]. They are not soluble
in water as a result of strong hydrophobic interactions
and the presence of disulfide bonds, only in aqueous
alcohol [6, 7].
Gliadin proteins are divided into four groups (α,
β, γ, and ω gliadins) on the basis of mobility in acidic
conditions of acid polyacrylamide gel electrophoresis
(A-PAGE). Some recent research on amino acid
sequences refer α and β gliadins to the same group (α/β)
[8, 9]. By amino acid sequences (complete and partial),
amino acid composition, and molecular weight, gliadins
are divided into: ω5, ω1,2, α+β, and γ gliadins [10, 11].
As for ω gliadins, they have a high content of glutamine,
proline, and phenylalanine. They are divided into ω5
(≈ 50 000 Da) and ω1.2 gliadins (≈ 40 000 Da).
In α+β and γ gliadins, the content of glutamine and
proline is much lower than in ω gliadins. The molecular
weights of these fractions overlap (≈ 28 000–35 000 Da).
They differ in the content of several amino acids
(tyrosine). Both fractions contain the N- and C-terminal
regions [12, 13].
Although the content of total gliadin proteins
depends on the type of wheat and growth conditions
(soil, climate, fertilization, etc.), α+β and γ gliadins are
the largest components, while ω gliadins are present in
smaller amounts [14, 15].
Research Article https://doi.org/10.21603/2308-4057-2021-2-364-370
Open Access Available online at http://jfrm.ru/en
Gliadin proteins from wheat flour:
the optimal determination conditions by ELISA
Željka Marjanović-Balaban1, Vesna Gojković Cvjetković2,* , Radoslav Grujić3
1 University of Banja Luka , Banja Luka, Bosnia and Herzegovina
2 University of East Sarajevo , East Sarajevo, Bosnia and Herzegovina
3 State High School of Medical Science, Prijedor, Bosnia and Herzegovina
* e-mail: vesna.gojkovic@yahoo.com
Received June 13, 2021; Accepted in revised form July 08, 2021; Published online X X, 2021
Abstract:
Introduction. The number of people with celiac disease is rapidly increasing. Gluten, is one of the most common food allergens,
consists of two fractions: gliadins and glutenins. The research objective was to determine the optimal conditions for estimating
gliadins by using enzyme-linked immunosorbent assay (ELISA).
Study objects and methods. The experiment involved wheat flour samples (0.10; 0.20, 0.25, 0.50, and 1.0 g) suspended in different
solvents (ethanol, methanol, 1-propanol, and isopropanol) of different concentrations (40, 50, 60, 70, 80, and 90% v/v). The samples
were diluted with Tris buffer in ratios of 1:50, 1:100, 1:150, and 1:200. The gliadin test was performed using a Gliadin/Gluten Biotech
commercial ELISA kit (Immunolab).
Results and discussion. The optimal conditions for determining gliadin proteins that provided the highest gliadin concentration were:
solvent – 70% v/v ethanol, extract:Tris buffer ratio – 1:50, and sample weight – 1.0 g.
Conclusion. The obtained results can be of great importance to determine gliadin/gluten concentrations in food products by rapid
analysis methods.
Keywords: Extraction, gluten, gliadins, wheat flour, enzyme-linked immunosorbent assay (ELISA)
Please cite this article in press as: Marjanović-Balaban Ž, Gojković Cvjetković V, Grujić R. Gliadin proteins from wheat flour: the
optimal determination conditions by ELISA. Foods and Raw Materials. 2021;9(2):364–-370. https://doi.org/10.21603/2308-4057-
2021-2-364-370.
Copyright © 2021, Marjanović-Balaban et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and
to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.
Foods and Raw Materials, 2021, vol. 9, no. 2
E-ISSN 2310-9599
ISSN 2308-4057
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Marjanović-Balaban Ž. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 364–370
Gluten is a common concern for people around the
world, especially in the United States, where nearly onethird
of the population have to reduce the intake of this
protein. Numerous studies have been conducted on the
adverse reactions of gluten and its impact on the health
of certain population groups [16–18].
Considering that the number of people with gluten
intolerance has been increasing in the last decade,
the research objective was to examine the optimal
conditions for determining the concentration of gliadin
by a rapid enzyme-linked immunosorbent assay method
(ELISA).
STUDY OBJECTS AND METHODS
The research featured wheat flour type 500 samples
with maximal ash content – 0.55%, maximal moisture –
15%, maximal acidity – 3, and protein content –
9.8 g/100 g. The samples were purchased on the market
of the Republic of Srpska, Bosnia and Herzegovina.
The gliadin test involved the following chemicals:
ethanol (Refined REAHEM, 96% v/v ethyl alcohol,
Srbobran), methanol (Lach-Ner, Czech Republic, high
purity, ≥ 99.99%), 1-propanol Lach-Ner, Czech Republic,
high purity, ≥ 99.00%), and isopropanol (Lach-Ner,
Czech Republic, high purity, 99.90%). The deionized
water was obtained in laboratory conditions using a
Water Technologies device W3T199551 (Siemens Ultra
Clear) at a conductivity of 0.055 mS/cm and temperature
of 20°C.
The commercial kit (Immunolab, GmbH, Gliadin/
Gluten ELISA, D-Kassel, Germany) contained the
following chemicals: a series of gliadin standard
solutions (concentrations 0, 2, 6, 20, and 60 ppm),
a conjugate (anti-gliadin peroxidase), a substrate
(tetramethylbenzidine, TMB), a stop solution (0.5 M
H2SO4), a buffer (Tris), and a wash solution (PBS +
Tween 20), plus 96 wells. According to the
manufacturer’s instructions, the putty is to be stored in
the refrigerator at 2–8°C.
Sample preparation. The wheat flour samples (1.0,
0.5, 0.25, 0.20, 0.10 g ± 0.0001 g) were suspended in
10.0 ml of solvent (ethanol, methanol, isopropanol, and
1-propanol) of different concentrations (40, 50, 60, 70,
80, and 90% v/v). The samples were homogenized with
an Ultra-Turrax homogenizer (IKA T25 digital, 10 000
rpm) for 5 min. The samples were then centrifuged
(Hettich zentrifugen, rotina 380 R) at 2000 rpm for
10 min. After centrifugation, the supernatant was
drained and diluted in a ratio of 1:50 with 10x
concentrated Tris buffer, which had been diluted
before use.
Determination gliadin proteins by ELISA.
The samples and 100 μL of gliadin standard solution
(concentrations 0, 2, 6, 20, and 60 ppm) were pipetted
into wells, followed by incubation for 20 min at room
temperature. The rinsing solution was concentrated
(10x) and diluted 1:9 with distilled water. The wells were
rinsed with 300 μL of the rinsing solution by adding it
into the wells; the procedure was repeated three times.
After washing, 100 μL of the conjugate (anti-gliadin
peroxidase) was pipetted into the wells and incubated
for 20 min. Then, the washing procedure was repeated,
and 100 μL of the substrate was put into the wells. To
react, they were left in a dark place for 20 min at 20°C
until the content of the well turned blue. Upon adding
100 μL of the stop solution (0.5 M H2SO4), the blue
color turned yellow. After mixing, the absorbance was
measured using an ELISA reader (Chromate, Awarenes
Technology) at 450 nm. The color was stable after
30 min.
RESULTS AND DISCUSSION
Table 1 shows the absorbance of the gliadin standard
solutions at the concentrations of 0, 2, 6, 20, and
60 ppm at a wavelength of 450 nm. The results made it
possible to calculate the dependence of the absorbance
on the protein solution concentration, as illustrated
by the calibration curve (Graph 1). The correlation
coefficient (R2 = 0.9997) showed a high dependence of
the absorbance on the concentration of standard gliadin
solutions.
Table 2 shows descriptive indicators of gliadin
concentration (ppm) values in extracts obtained from
wheat flour samples after extraction with different
concentrations of ethanol. During the extraction, which
lasted for 20 min, the samples were mixed after every
Table 1 Absorption of gliadin standard solutions at 450 nm
Concentration of gliadin standard solutions, ppm 0 2 6 20 60
Absorbance (450 nm) 0.208 ± 0.02 0.365 ± 0.04 0.598 ± 0.01 1.421 ± 0.08 2.588 ± 0.17
Figure 1 Dependence of absorbance on the concentration
of gliadin standard solutions
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5 min. The obtained extracts were diluted with Tris
buffer in a ratio of 1:50.
A descriptive analysis showed that the highest
gliadin concentration was obtained after extraction
with 70% ethanol (104.15 ppm). Extraction with 90%
ethanol demonstrated the lowest gliadin concentration
(69.47 ppm). A one-factor analysis of variance of
different groups revealed a statistically significant
difference in the gliadin concentration at F(5.30) =
137.58 and Sig. = 0.000.
Table 2 shows that the increased solvent
concentration between 40 and 70% affected the
efficiency of gliadin protein extraction from wheat flour
samples: the protein concentration increased. However,
a further increase in the solvent concentration (80 and
90%) reduced the extraction efficiency: gliadin protein
concentration was lower than in the case of 70% ethanol.
Table 3 illustrates the descriptive indicators of
gliadin concentration (ppm) after extraction with
methanol of different concentrations.
The highest concentration of gliadins was obtained
after extraction with 70% methanol (95.49 ppm),
while 80% methanol showed the lowest concentration
(73.77 ppm). A one-factor analysis of variance of
different groups showed a statistically significant
difference in the gliadin concentrations at F(5.30) =
44.48 and Sig. = 0.000 (Table 3).
Under these conditions, the protein extraction was
more effective when the methanol concentration was
40%-70%, while a further increase in the concentration
of methanol (80 and 90%) reduced the extraction
efficiency.
Table 2 Descriptive indicators of gliadins measured in wheat flour extracts at different solvent concentrations
(sample weight 1.0 g ± 0.0001, solvent ethanol)
Ethanol
concentration
N Xav SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
40% 6 85.42 3.40 1.39 81.86 88.99 78.68 87.61
50% 6 88.83 3.33 1.36 85.34 92.32 83.66 93.38
60% 6 102.23 2.65 1.08 99.44 105.02 98.71 105.21
70% 6 104.15 2.06 0.84 101.99 106.32 100.93 107.21
80% 6 75.74 1.63 0.67 74.03 77.45 73.67 78.41
90% 6 69.47 3.72 1.52 65.56 73.38 62.92 73.69
ANOVA F(5.30) = 137.58, Sig. = 0.000, eta square = 5781.29/6033.41 = 0.96
Table 3 Descriptive indicators of gliadins in wheat flour extracts at different solvent concentrations (sample weight 1.0 g ± 0.0001,
solvent methanol)
Methanol
concentration
N Xav SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
40% 6 83.29 4.85 1.98 78.20 88.38 74.13 88.26
50% 6 88.70 2.02 0.83 86.58 90.83 86.65 91.81
60% 6 89.51 3.26 1.33 86.09 92.93 83.98 93.41
70% 6 95.49 2.69 1.10 92.67 98.31 91.23 99.33
80% 6 73.77 2.81 1.15 70.83 76.72 70.52 78.03
90% 6 73.81 3.22 1.31 70.43 77.18 69.04 78.33
ANOVA F(5.30) = 44.48, Sig. = 0.000, eta square = 2360.62/2679.04 = 0.88
Table 4 Descriptive indicators of gliadins in wheat flour extracts at different solvent concentrations (sample weight 1.0 g ± 0.0001,
solvent 1-propanol)
1-propanol
concentration
N Xav SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
40% 6 97.36 1.92 0.78 95.35 99.37 93.59 98.90
50% 6 98.40 1.99 0.82 96.30 100.49 95.57 100.98
60% 6 101.16 2.01 0.82 99.05 103.27 97.70 103.06
70% 6 94.33 1.91 0.78 92.32 96.33 91.19 96.79
80% 6 96.40 1.88 0.77 94.43 98.37 93.38 98.70
90% 6 84.97 1.75 0.72 83.13 86.81 83.29 88.18
ANOVA F(5.30) = 51.45, Sig. = 0.000, eta square = 941.55/1051.34 = 0.89
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Table 4 shows the descriptive indicators of gliadin
concentrations (ppm) after extraction with 1-propanol of
different concentrations.
The highest concentration of gliadins was obtained
after extraction with 60% 1-propanol (101.16 ppm), while
90% 1-propanol resulted in the lowest concentration
(84.97 ppm). A one-factor analysis of variance of
different groups revealed a statistically significant
difference in the gliadin concentration at F(5.30) = 51.45
and Sig. = 0.000 (Table 4).
A lower solvent concentration of 1-propanol between
40 and 60% increased the efficiency of gliadin protein
extraction, while the protein extraction efficiency
tended to decrease with a further increase in solvent
concentration (70, 80 and 90 %), i.e. the concentration
decreased.
Table 5 shows the descriptive indicators of gliadin
concentrations (ppm) after extraction with isopropanol
of different concentrations.
The highest concentration of gliadin was obtained
after extraction with 70% isopropanol (103.35 ppm).
Extraction with 40% isopropanol showed the lowest
concentration of gliadins (83.65 ppm). A one-factor
analysis of variance showed a statistically significant
difference in gliadin concentrations at F(5.30) = 14.72
and Sig. = 0.000 (Table 5).
A higher solvent concentration of isopropanol for
gliadin protein extraction between 40 and 70% increased
the extraction efficiency, while further increase in the
solvent concentration (80 and 90%) resulted in a lower
extraction efficiency, compared to the experiment with
70% isopropanol.
Based on Tables 2–5, the best efficiency of gliadin
protein extraction was achieved during the experiments
with 70% ethanol and 70% isopropanol as solvents.
Table 6 demonstrates the descriptive indicators of the
gliadin concentration (ppm) after extraction with 70%
ethanol, followed by dilution of the extract with different
Tris buffer concentrations.
The extract:Tris buffer ratios of 1:50 and 1:200
demonatrsted the highest and the lowest concentration
of gliadins (104.15 and 84.35 ppm, respectively). A
one-factor analysis of variance of different groups
showed a statistically significant difference in gliadin
concentration a t F (3.20) = 8 0.62 a nd S ig. = 0 .000. A n
increase in Tris buffer concentration decreased gliadins.
Table 5 Descriptive indicators of gliadins in wheat flour extracts at different solvent concentrations (sample weight 1.0 g ± 0.0001,
solvent isopropanol)
Isopropanol
concentration
N Xav SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
40% 6 83.65 7.63 3.12 75.64 91.66 73.18 92.36
50% 6 92.77 3.80 1.55 88.79 96.75 86.35 97.22
60% 6 92.27 3.72 1.52 88.36 96.18 87.31 97.97
70% 6 103.35 2.97 1.21 100.23 106.46 98.81 107.23
80% 6 93.29 3.69 1.51 89.42 97.17 86.45 97.27
90% 6 85.24 3.38 1.38 81.69 88.79 78.80 88.75
ANOVA F(5.30) = 14.72, Sig. = 0.000, eta square = 1476.98/2079.01 = 0.71
Table 6 Descriptive indicators of gliadins in wheat flour extracts diluted with different Tris buffer concentrations
(sample weight 1.0 g ± 0.0001, solvent 70% ethanol)
Extract:Tris
buffer ratio
N Xav SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
1:50 6 104.15 2.06 0.84 101.99 106.32 100.93 107.21
1:100 6 95.08 0.96 0.39 94.07 96.08 93.29 95.89
1:150 6 89.06 2.88 1.18 86.04 92.09 83.68 91.27
1:200 6 84.35 2.87 1.17 81.33 87.36 79.48 87.88
ANOVA F(3.20) = 80.62, Sig. = 0.000, eta square = 1314.04/1422.70 = 0.92
Table 7 Descriptive indicators of gliadins in wheat flour extracts diluted with different Tris buffer concentrations
(sample weight 1.0 g ± 0.0001, solvent 70% isopropanol)
Extract:Tris
buffer ratio
N Xav SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
1:50 6 103.35 2.97 1.21 100.23 106.46 98.81 107.23
1:100 6 84.87 1.47 0.60 83.33 86.42 83.69 87.69
1:150 6 74.24 2.23 0.91 71.89 76.58 70.40 76.84
1:200 6 65.95 3.25 1.33 62.53 69.36 61.12 70.30
ANOVA F(3.20) = 235.73, Sig. = 0.000, eta square = 4691.03/4823.70 = 0.97
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Table 7 shows the descriptive indicators of gliadin
concentrations (ppm) in wheat flour extracts obtained
after extraction with 70% isopropanol and diluted with
different Tris buffer concentrations.
The highest concentration of gliadins was
obtained in the extract diluted with Tris buffer
in a ratio of 1:50 (103.35 ppm). The ratio of 1:200
showed the lowest concentration of gliadins
(65.95 ppm). A one-factor analysis of variance
of different groups demonstrated a statistically
significant difference in the concentration of gliadins
calculated by the eta square indicator at F(3.20) =
235.73 and Sig. = 0.000 (Table 7). An increase in Tris
buffer decreased gliadin protein concentration.
Table 8 shows the descriptive indicators of gliadins
(ppm) extracted from wheat flour samples of different
weights with 70% ethanol as solvent. The extracts were
diluted with Tris buffer in a ratio of 1:50.
The highest and lowest concentration of gliadins
was observed in samples with wheat flour weights of
1.00 g and 0.10 g (104.15 and 48.41 ppm, respectively).
A one-factor analysis of variance of different groups
showed a statistically significant difference in gliadin
concentration a t F (4.25) = 2 0.85 a nd S ig. = 0 .000
(Table 8).
Table 9 shows descriptive indicators of gliadins
(ppm) extracted from wheat flour samples of different
weights with 70% isopropanol as solvent. The extracts
were diluted with Tris buffer in a ratio of 1:50.
Samples with wheat flour weights of 1.00 and
0.10 g had the highest and the lowest gliadin concentrations
(103.35 and 53.59 ppm, respectively). A onefactor
analysis of variance of different groups showed a
statistically significant difference in gliadin concentration
a t F (4.25) = 4 4.05 a nd S ig. = 0 .000 ( Table 9 ). A n
increase in the weight of the wheat flour increased the
gliadin protein concentration value.
Ayob et al. developed an enzyme-linked immunosorbent
assay (ELISA) in order to determine gliadin
proteins in food [19]. They studied three gliadins
extracted from wheat flour samples with 70% (v/v)
ethanol. The samples were vortexed for 30 min. Prior
to the analysis, they were diluted with water in different
ratios (1:10, 1:100, 1:1000, and 1:10 000). The highest
concentration of gliadin was obtained in the sample
diluted 1:10, and the lowest – in the sample diluted
1:10 000.
Allred and Ritter determined the gliadin and
glutenin content in flour and in products available on the
market, using four commercial ELISA tests [20]. They
extracted gliadin with 0.3 M Na-iodide and 7.5% (v/v)
1-propanol. The first test detected gluten in 29 out of
40 analyzed products, the second – in 20 products, the
third – in 12 products, and the fourth in 18 products.
Gujral et al. determined the gliadin content by
ELISA sandwich technique [21]. Gliadins were extracted
with 250 mM 2-mercaptoethanol+2M guanidine
hydrochloride. The scientists added 7.5 mL of 80% (v/v)
ethanol to the solution. Vortex mixing was performed
for 30 min. The gliadin content in wheat flour was
7.4 μg/kg.
The results obtained in this work are in conformity
with the research by Ayob et al., who also extracted
gliadins with 70% (v/v) ethanol and detected the
dependance beteween an increasing dilution and a
lowering gliadin concentration [19].
Table 8 Descriptive indicators of gliadins in wheat flour extracts at different sample weights (solvent 70% ethanol, extract:buffer
ratio 1:50)
Sample
weight, g
N Xav SD Std.
error
95% confidence interval of average Min Max
Lower bound Upper bound
0.10 ± 0.0001 6 48.41 1.06 0.43 47.30 49.53 46.69 49.73
0.20 ± 0.0001 6 54.67 4.40 1.80 50.05 59.28 51.51 63.30
0.25 ± 0.0001 6 55.80 3.62 1.48 52.01 59.60 52.13 61.72
0.50 ± 0.0001 6 63.94 3.64 1.49 60.12 67.77 60.37 68.91
1.00 ± 0.0001 6 104.15 2.06 0.84 101.99 106.32 100.93 107.21
ANOVA F(4.25) = 20.85, Sig. = 0.000, eta square = 732.45/966.64 = 0.76
Table 9 Descriptive indicators of gliadins in wheat flour extracts at different sample weights (solvent 70% isopropanol,
extract:buffer ratio 1:50)
Sample
weight, g
N Xav SD Std. error 95% confidence interval of average Min Max
Lower Bound Upper bound
0.10 ± 0.0001 6 53.59 1.58 0.65 51.93 55.25 51.81 56.14
0.20 ± 0.0001 6 54.96 2.98 1.22 51.84 58.09 52.18 60.33
0.25 ± 0.0001 6 58.77 1.66 0.68 57.02 60.51 56.24 61.12
0.50 ± 0.0001 6 65.58 1.25 0.51 64.27 66.88 64.22 67.79
1.00 ± 0.0001 6 103.35 2.97 1.21 100.23 106.46 98.81 107.23
ANOVA F(4.25) = 44.05, Sig. = 0.000, eta square = 518.69/597.20 = 0.87
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CONCLUSION
To determine the optimal conditions for estimating
gliadin proteins by the ELISA method, we used
different solvents (ethanol, methanol, 1-propanol, and
isopropanol) at different concentrations (40, 50, 60, 70,
80, and 90%) as well as varied wheat flour weights (0.10,
0.20, 0.25, 0.50 and 1.00 g) and extract:buffer ratios
(1:50, 1:100, 1:150, and 1:200).
The experiments demonstrated that 70% ethanol
and 70% isopropanol were the optimal solvents,
which resulted in the highest gliadin concentrations.
However, 70% ethanol had a better financial feasibility.
70% ethanol, a Tris buffer dilution ratio of 1:50, and a
wheat flour sample weight of 1.00 g were the optimal
conditions that provided the highest concentration of
gliadins (104.15 ppm).
Considering the growing number of people with
celiac disease, the results obtained can be of great
fundamental importance in the study and determination
of gliadin/gluten concentrations in food products labeled
as gluten or gluten free by ELISA rapid method.
CONTRIBUTION
Ž. Marjanović-Balaban, V. Gojković Cvjetković,
R. Grujić conceived, designed, and performed the
experiments, analyzed the data, contributed reagents,
materials and analytical tools, and wrote the paper.
CONFLICT OF INTEREST
The authors declare no potential conflict of interests
regarding the publication of this article.
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