INTRODUCTIONGluten is the one of the most common food allergens.According to the Codex Alimentarius [1], gluten isdefined as a protein fraction of wheat, rye, barley, oats,their cross varieties, and derivatives, which some peopleare sensitive to [2]. Gliadins and glutenins are twofractions present in approximately equal amounts ingluten [3].Gliadins are respresented by monomers. Due to thehigh content of glutamine and proline, these proteinsare also called “prolamins” [4, 5]. They are not solublein water as a result of strong hydrophobic interactionsand the presence of disulfide bonds, only in aqueousalcohol [6, 7].Gliadin proteins are divided into four groups (α,β, γ, and ω gliadins) on the basis of mobility in acidicconditions of acid polyacrylamide gel electrophoresis(A-PAGE). Some recent research on amino acidsequences 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, gliadinsare 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 andproline is much lower than in ω gliadins. The molecularweights 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-terminalregions [12, 13].Although the content of total gliadin proteinsdepends on the type of wheat and growth conditions(soil, climate, fertilization, etc.), α+β and γ gliadins arethe largest components, while ω gliadins are present insmaller amounts [14, 15].Research Article https://doi.org/10.21603/2308-4057-2021-2-364-370Open Access Available online at http://jfrm.ru/enGliadin proteins from wheat flour:the optimal determination conditions by ELISAŽeljka Marjanović-Balaban1, Vesna Gojković Cvjetković2,* , Radoslav Grujić31 University of Banja Luka , Banja Luka, Bosnia and Herzegovina2 University of East Sarajevo , East Sarajevo, Bosnia and Herzegovina3 State High School of Medical Science, Prijedor, Bosnia and Herzegovina* e-mail: vesna.gojkovic@yahoo.comReceived June 13, 2021; Accepted in revised form July 08, 2021; Published online X X, 2021Abstract: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 estimatinggliadins 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 differentsolvents (ethanol, methanol, 1-propanol, and isopropanol) of different concentrations (40, 50, 60, 70, 80, and 90% v/v). The sampleswere 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 Biotechcommercial 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 rapidanalysis 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: theoptimal 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 InternationalLicense (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format andto 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. 2E-ISSN 2310-9599ISSN 2308-4057365Marjanović-Balaban Ž. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 364–370Gluten is a common concern for people around theworld, especially in the United States, where nearly onethirdof the population have to reduce the intake of thisprotein. Numerous studies have been conducted on theadverse reactions of gluten and its impact on the healthof certain population groups [16–18].Considering that the number of people with glutenintolerance has been increasing in the last decade,the research objective was to examine the optimalconditions for determining the concentration of gliadinby a rapid enzyme-linked immunosorbent assay method(ELISA).STUDY OBJECTS AND METHODSThe research featured wheat flour type 500 sampleswith 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 marketof 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, highpurity, ≥ 99.99%), 1-propanol Lach-Ner, Czech Republic,high purity, ≥ 99.00%), and isopropanol (Lach-Ner,Czech Republic, high purity, 99.90%). The deionizedwater was obtained in laboratory conditions using aWater Technologies device W3T199551 (Siemens UltraClear) at a conductivity of 0.055 mS/cm and temperatureof 20°C.The commercial kit (Immunolab, GmbH, Gliadin/Gluten ELISA, D-Kassel, Germany) contained thefollowing chemicals: a series of gliadin standardsolutions (concentrations 0, 2, 6, 20, and 60 ppm),a conjugate (anti-gliadin peroxidase), a substrate(tetramethylbenzidine, TMB), a stop solution (0.5 MH2SO4), a buffer (Tris), and a wash solution (PBS +Tween 20), plus 96 wells. According to themanufacturer’s instructions, the putty is to be stored inthe 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 in10.0 ml of solvent (ethanol, methanol, isopropanol, and1-propanol) of different concentrations (40, 50, 60, 70,80, and 90% v/v). The samples were homogenized withan Ultra-Turrax homogenizer (IKA T25 digital, 10 000rpm) for 5 min. The samples were then centrifuged(Hettich zentrifugen, rotina 380 R) at 2000 rpm for10 min. After centrifugation, the supernatant wasdrained and diluted in a ratio of 1:50 with 10xconcentrated Tris buffer, which had been dilutedbefore use.Determination gliadin proteins by ELISA.The samples and 100 μL of gliadin standard solution(concentrations 0, 2, 6, 20, and 60 ppm) were pipettedinto wells, followed by incubation for 20 min at roomtemperature. The rinsing solution was concentrated(10x) and diluted 1:9 with distilled water. The wells wererinsed with 300 μL of the rinsing solution by adding itinto the wells; the procedure was repeated three times.After washing, 100 μL of the conjugate (anti-gliadinperoxidase) was pipetted into the wells and incubatedfor 20 min. Then, the washing procedure was repeated,and 100 μL of the substrate was put into the wells. Toreact, they were left in a dark place for 20 min at 20°Cuntil the content of the well turned blue. Upon adding100 μL of the stop solution (0.5 M H2SO4), the bluecolor turned yellow. After mixing, the absorbance wasmeasured using an ELISA reader (Chromate, AwarenesTechnology) at 450 nm. The color was stable after30 min.RESULTS AND DISCUSSIONTable 1 shows the absorbance of the gliadin standardsolutions at the concentrations of 0, 2, 6, 20, and60 ppm at a wavelength of 450 nm. The results made itpossible to calculate the dependence of the absorbanceon the protein solution concentration, as illustratedby the calibration curve (Graph 1). The correlationcoefficient (R2 = 0.9997) showed a high dependence ofthe absorbance on the concentration of standard gliadinsolutions.Table 2 shows descriptive indicators of gliadinconcentration (ppm) values in extracts obtained fromwheat flour samples after extraction with differentconcentrations of ethanol. During the extraction, whichlasted for 20 min, the samples were mixed after everyTable 1 Absorption of gliadin standard solutions at 450 nmConcentration of gliadin standard solutions, ppm 0 2 6 20 60Absorbance (450 nm) 0.208 ± 0.02 0.365 ± 0.04 0.598 ± 0.01 1.421 ± 0.08 2.588 ± 0.17Figure 1 Dependence of absorbance on the concentrationof gliadin standard solutions366Marjanović-Balaban Ž. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 364–3705 min. The obtained extracts were diluted with Trisbuffer in a ratio of 1:50.A descriptive analysis showed that the highestgliadin concentration was obtained after extractionwith 70% ethanol (104.15 ppm). Extraction with 90%ethanol demonstrated the lowest gliadin concentration(69.47 ppm). A one-factor analysis of variance ofdifferent groups revealed a statistically significantdifference in the gliadin concentration at F(5.30) =137.58 and Sig. = 0.000.Table 2 shows that the increased solventconcentration between 40 and 70% affected theefficiency of gliadin protein extraction from wheat floursamples: the protein concentration increased. However,a further increase in the solvent concentration (80 and90%) reduced the extraction efficiency: gliadin proteinconcentration was lower than in the case of 70% ethanol.Table 3 illustrates the descriptive indicators ofgliadin concentration (ppm) after extraction withmethanol of different concentrations.The highest concentration of gliadins was obtainedafter extraction with 70% methanol (95.49 ppm),while 80% methanol showed the lowest concentration(73.77 ppm). A one-factor analysis of variance ofdifferent groups showed a statistically significantdifference in the gliadin concentrations at F(5.30) =44.48 and Sig. = 0.000 (Table 3).Under these conditions, the protein extraction wasmore effective when the methanol concentration was40%-70%, while a further increase in the concentrationof methanol (80 and 90%) reduced the extractionefficiency.Table 2 Descriptive indicators of gliadins measured in wheat flour extracts at different solvent concentrations(sample weight 1.0 g ± 0.0001, solvent ethanol)EthanolconcentrationN Xav SD Std.error95% confidence interval of average Min MaxLower bound Upper bound40% 6 85.42 3.40 1.39 81.86 88.99 78.68 87.6150% 6 88.83 3.33 1.36 85.34 92.32 83.66 93.3860% 6 102.23 2.65 1.08 99.44 105.02 98.71 105.2170% 6 104.15 2.06 0.84 101.99 106.32 100.93 107.2180% 6 75.74 1.63 0.67 74.03 77.45 73.67 78.4190% 6 69.47 3.72 1.52 65.56 73.38 62.92 73.69ANOVA F(5.30) = 137.58, Sig. = 0.000, eta square = 5781.29/6033.41 = 0.96Table 3 Descriptive indicators of gliadins in wheat flour extracts at different solvent concentrations (sample weight 1.0 g ± 0.0001,solvent methanol)MethanolconcentrationN Xav SD Std.error95% confidence interval of average Min MaxLower bound Upper bound40% 6 83.29 4.85 1.98 78.20 88.38 74.13 88.2650% 6 88.70 2.02 0.83 86.58 90.83 86.65 91.8160% 6 89.51 3.26 1.33 86.09 92.93 83.98 93.4170% 6 95.49 2.69 1.10 92.67 98.31 91.23 99.3380% 6 73.77 2.81 1.15 70.83 76.72 70.52 78.0390% 6 73.81 3.22 1.31 70.43 77.18 69.04 78.33ANOVA F(5.30) = 44.48, Sig. = 0.000, eta square = 2360.62/2679.04 = 0.88Table 4 Descriptive indicators of gliadins in wheat flour extracts at different solvent concentrations (sample weight 1.0 g ± 0.0001,solvent 1-propanol)1-propanolconcentrationN Xav SD Std.error95% confidence interval of average Min MaxLower bound Upper bound40% 6 97.36 1.92 0.78 95.35 99.37 93.59 98.9050% 6 98.40 1.99 0.82 96.30 100.49 95.57 100.9860% 6 101.16 2.01 0.82 99.05 103.27 97.70 103.0670% 6 94.33 1.91 0.78 92.32 96.33 91.19 96.7980% 6 96.40 1.88 0.77 94.43 98.37 93.38 98.7090% 6 84.97 1.75 0.72 83.13 86.81 83.29 88.18ANOVA F(5.30) = 51.45, Sig. = 0.000, eta square = 941.55/1051.34 = 0.89367Marjanović-Balaban Ž. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 364–370Table 4 shows the descriptive indicators of gliadinconcentrations (ppm) after extraction with 1-propanol ofdifferent concentrations.The highest concentration of gliadins was obtainedafter extraction with 60% 1-propanol (101.16 ppm), while90% 1-propanol resulted in the lowest concentration(84.97 ppm). A one-factor analysis of variance ofdifferent groups revealed a statistically significantdifference in the gliadin concentration at F(5.30) = 51.45and Sig. = 0.000 (Table 4).A lower solvent concentration of 1-propanol between40 and 60% increased the efficiency of gliadin proteinextraction, while the protein extraction efficiencytended to decrease with a further increase in solventconcentration (70, 80 and 90 %), i.e. the concentrationdecreased.Table 5 shows the descriptive indicators of gliadinconcentrations (ppm) after extraction with isopropanolof different concentrations.The highest concentration of gliadin was obtainedafter extraction with 70% isopropanol (103.35 ppm).Extraction with 40% isopropanol showed the lowestconcentration of gliadins (83.65 ppm). A one-factoranalysis of variance showed a statistically significantdifference in gliadin concentrations at F(5.30) = 14.72and Sig. = 0.000 (Table 5).A higher solvent concentration of isopropanol forgliadin protein extraction between 40 and 70% increasedthe extraction efficiency, while further increase in thesolvent concentration (80 and 90%) resulted in a lowerextraction efficiency, compared to the experiment with70% isopropanol.Based on Tables 2–5, the best efficiency of gliadinprotein extraction was achieved during the experimentswith 70% ethanol and 70% isopropanol as solvents.Table 6 demonstrates the descriptive indicators of thegliadin concentration (ppm) after extraction with 70%ethanol, followed by dilution of the extract with differentTris buffer concentrations.The extract:Tris buffer ratios of 1:50 and 1:200demonatrsted the highest and the lowest concentrationof gliadins (104.15 and 84.35 ppm, respectively). Aone-factor analysis of variance of different groupsshowed a statistically significant difference in gliadinconcentration a t F (3.20) = 8 0.62 a nd S ig. = 0 .000. A nincrease 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)IsopropanolconcentrationN Xav SD Std.error95% confidence interval of average Min MaxLower bound Upper bound40% 6 83.65 7.63 3.12 75.64 91.66 73.18 92.3650% 6 92.77 3.80 1.55 88.79 96.75 86.35 97.2260% 6 92.27 3.72 1.52 88.36 96.18 87.31 97.9770% 6 103.35 2.97 1.21 100.23 106.46 98.81 107.2380% 6 93.29 3.69 1.51 89.42 97.17 86.45 97.2790% 6 85.24 3.38 1.38 81.69 88.79 78.80 88.75ANOVA F(5.30) = 14.72, Sig. = 0.000, eta square = 1476.98/2079.01 = 0.71Table 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:Trisbuffer ratioN Xav SD Std.error95% confidence interval of average Min MaxLower bound Upper bound1:50 6 104.15 2.06 0.84 101.99 106.32 100.93 107.211:100 6 95.08 0.96 0.39 94.07 96.08 93.29 95.891:150 6 89.06 2.88 1.18 86.04 92.09 83.68 91.271:200 6 84.35 2.87 1.17 81.33 87.36 79.48 87.88ANOVA F(3.20) = 80.62, Sig. = 0.000, eta square = 1314.04/1422.70 = 0.92Table 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:Trisbuffer ratioN Xav SD Std.error95% confidence interval of average Min MaxLower bound Upper bound1:50 6 103.35 2.97 1.21 100.23 106.46 98.81 107.231:100 6 84.87 1.47 0.60 83.33 86.42 83.69 87.691:150 6 74.24 2.23 0.91 71.89 76.58 70.40 76.841:200 6 65.95 3.25 1.33 62.53 69.36 61.12 70.30ANOVA F(3.20) = 235.73, Sig. = 0.000, eta square = 4691.03/4823.70 = 0.97368Marjanović-Balaban Ž. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 364–370Table 7 shows the descriptive indicators of gliadinconcentrations (ppm) in wheat flour extracts obtainedafter extraction with 70% isopropanol and diluted withdifferent Tris buffer concentrations.The highest concentration of gliadins wasobtained in the extract diluted with Tris bufferin a ratio of 1:50 (103.35 ppm). The ratio of 1:200showed the lowest concentration of gliadins(65.95 ppm). A one-factor analysis of varianceof different groups demonstrated a statisticallysignificant difference in the concentration of gliadinscalculated by the eta square indicator at F(3.20) =235.73 and Sig. = 0.000 (Table 7). An increase in Trisbuffer decreased gliadin protein concentration.Table 8 shows the descriptive indicators of gliadins(ppm) extracted from wheat flour samples of differentweights with 70% ethanol as solvent. The extracts werediluted with Tris buffer in a ratio of 1:50.The highest and lowest concentration of gliadinswas observed in samples with wheat flour weights of1.00 g and 0.10 g (104.15 and 48.41 ppm, respectively).A one-factor analysis of variance of different groupsshowed a statistically significant difference in gliadinconcentration 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 differentweights with 70% isopropanol as solvent. The extractswere diluted with Tris buffer in a ratio of 1:50.Samples with wheat flour weights of 1.00 and0.10 g had the highest and the lowest gliadin concentrations(103.35 and 53.59 ppm, respectively). A onefactoranalysis of variance of different groups showed astatistically significant difference in gliadin concentrationa t F (4.25) = 4 4.05 a nd S ig. = 0 .000 ( Table 9 ). A nincrease in the weight of the wheat flour increased thegliadin protein concentration value.Ayob et al. developed an enzyme-linked immunosorbentassay (ELISA) in order to determine gliadinproteins in food [19]. They studied three gliadinsextracted from wheat flour samples with 70% (v/v)ethanol. The samples were vortexed for 30 min. Priorto the analysis, they were diluted with water in differentratios (1:10, 1:100, 1:1000, and 1:10 000). The highestconcentration of gliadin was obtained in the samplediluted 1:10, and the lowest – in the sample diluted1:10 000.Allred and Ritter determined the gliadin andglutenin content in flour and in products available on themarket, using four commercial ELISA tests [20]. Theyextracted gliadin with 0.3 M Na-iodide and 7.5% (v/v)1-propanol. The first test detected gluten in 29 out of40 analyzed products, the second – in 20 products, thethird – in 12 products, and the fourth in 18 products.Gujral et al. determined the gliadin content byELISA sandwich technique [21]. Gliadins were extractedwith 250 mM 2-mercaptoethanol+2M guanidinehydrochloride. The scientists added 7.5 mL of 80% (v/v)ethanol to the solution. Vortex mixing was performedfor 30 min. The gliadin content in wheat flour was7.4 μg/kg.The results obtained in this work are in conformitywith the research by Ayob et al., who also extractedgliadins with 70% (v/v) ethanol and detected thedependance beteween an increasing dilution and alowering gliadin concentration [19].Table 8 Descriptive indicators of gliadins in wheat flour extracts at different sample weights (solvent 70% ethanol, extract:bufferratio 1:50)Sampleweight, gN Xav SD Std.error95% confidence interval of average Min MaxLower bound Upper bound0.10 ± 0.0001 6 48.41 1.06 0.43 47.30 49.53 46.69 49.730.20 ± 0.0001 6 54.67 4.40 1.80 50.05 59.28 51.51 63.300.25 ± 0.0001 6 55.80 3.62 1.48 52.01 59.60 52.13 61.720.50 ± 0.0001 6 63.94 3.64 1.49 60.12 67.77 60.37 68.911.00 ± 0.0001 6 104.15 2.06 0.84 101.99 106.32 100.93 107.21ANOVA F(4.25) = 20.85, Sig. = 0.000, eta square = 732.45/966.64 = 0.76Table 9 Descriptive indicators of gliadins in wheat flour extracts at different sample weights (solvent 70% isopropanol,extract:buffer ratio 1:50)Sampleweight, gN Xav SD Std. error 95% confidence interval of average Min MaxLower Bound Upper bound0.10 ± 0.0001 6 53.59 1.58 0.65 51.93 55.25 51.81 56.140.20 ± 0.0001 6 54.96 2.98 1.22 51.84 58.09 52.18 60.330.25 ± 0.0001 6 58.77 1.66 0.68 57.02 60.51 56.24 61.120.50 ± 0.0001 6 65.58 1.25 0.51 64.27 66.88 64.22 67.791.00 ± 0.0001 6 103.35 2.97 1.21 100.23 106.46 98.81 107.23ANOVA F(4.25) = 44.05, Sig. = 0.000, eta square = 518.69/597.20 = 0.87369Marjanović-Balaban Ž. et al. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 364–370CONCLUSIONTo determine the optimal conditions for estimatinggliadin proteins by the ELISA method, we useddifferent solvents (ethanol, methanol, 1-propanol, andisopropanol) 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% ethanoland 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 awheat flour sample weight of 1.00 g were the optimalconditions that provided the highest concentration ofgliadins (104.15 ppm).Considering the growing number of people withceliac disease, the results obtained can be of greatfundamental importance in the study and determinationof gliadin/gluten concentrations in food products labeledas gluten or gluten free by ELISA rapid method.CONTRIBUTIONŽ. Marjanović-Balaban, V. Gojković Cvjetković,R. Grujić conceived, designed, and performed theexperiments, analyzed the data, contributed reagents,materials and analytical tools, and wrote the paper.CONFLICT OF INTERESTThe authors declare no potential conflict of interestsregarding the publication of this article.