Performance assessment of a new G12/A1 antibody-based rapid ELISA using commercially available and gluten-spiked food samples

doi:10.21203/rs.3.rs-3345061/v1

Download PDF

Research Article

Performance assessment of a new G12/A1 antibody-based rapid ELISA using commercially available and gluten-spiked food samples

https://doi.org/10.21203/rs.3.rs-3345061/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Food products with < 20 mg/kg gluten can be labeled ‘gluten-free’ according to international regulations. Several antibodies based ELISAs have been develop to track gluten traces in food products. Among them, R5 and G12 antibody-based ELISAs are the frequently used methods. However, these antibodies have certain limitations.

Aims

We evaluated the accuracy of G12/A1 antibody-based 'Glutentox ELISA Rapid G12' and compared the results with the current reference method i.e., R5 antibody-based ‘Ridascreen R5 ELISA’.

Methods

In the first step, performance of Glutentox ELISA Rapid G12 kit was inspected by determination of the threshold value i.e.,> or < 20 mg/kg gluten in different food products. In the second step, quantification accuracy was assessed by quantification of gluten in gluten-free food products spiked with gliadin reference material.

Results

In total 47 food products (naturally and labeled gluten-free, and food with traces of gluten) were included. Of them, 29 products were quantified with < 20 mg/kg, and 18 with a low level of gluten by both the kits. Six out of 29 gluten-free products were used for the recovery test at different spike levels. Gluten concentration and mean recovery rates of individual kits showed consintency.

Conclusions

GlutenTox Rapid G12 ELISA could be an appropriate choice for detecting gluten in food products but needs more investigation e.g. more in-house validation and a collaborative test.

celiac disease

G12 ELISA

R5 ELISA

gluten

gluten contamination

gluten concentration

PWG-gliadin

spike and recovery

Celiac disease (CD) is an autoimmune condition that occurs in genetically predisposed individuals who develop an intense T-cell mediated immune response on exposure to the gluten protein found in wheat, barley, rye, and related grains [1]. Aprroximately 1% of the world's population is affected with CD [2]. A life-long complete elimination of gluten from the diet is the only accepted treatment for CD so far [3]. As wheat is a pervasive grain, complete elimination of gluten from the diet is difficult to achieve[4–6]. Some CD patients are highly sensitive to gluten traces (> 10 mg/daily) therefore, they must be protected from toxic gluten exposure [7]. According to the European Commission, Food and Drug Administration (FDA), and Codex Alimentarius, food products containing < 20 mg/kg of gluten can be labeled as ‘gluten-free’ and are safe for CD patients[8–10]. However, CD patients, similar to healthy individuals, are strongly dependent on commercially available food, and this is a problem for them since gluten contamination may occur along the food chain [6, 11, 12]. Therefore, an accurate gluten detection tool is needed to quantify the amount of gluten in gluten-free food products [13]. To track the minute amount of gluten in food, the Enzyme-Linked Immunosorbent Assay (ELISA) method is the most used and considered a ‘method of choice’[6, 14]. Different antibodies such as R5, G12, α-20, MIoBS, DQ2.5-glia-α3, and Skerritt have been developed to detect gluten traces in the food products [6, 15]. However, they have certain limitations. They often show significant discrepancies in the gluten estimation results, and the gluten extraction process varies among the ELISA kits. Different gluten ELISA methods use different calibration standards contributing the variability in the gluten quantity as not all commercial assays use Prolamin Working Group (PWG) gliadin standard (often for the economic reasons). Matrix interference, antibody-binding affinities, and extraction differences also contribute to variability among gluten ELISA methods. There are no official reference materials approved, and there is no universal unit of measurement of gluten [13, 16–18]. Therefore, the quest for an efficient method has not ended, and there is a need for an accurate method that can precisely determine the gluten content in all types of the food [13].

Among above mentioned antibodies, the R5 antibody-based gluten ELISA test kit i.e. RIDASCREEN^® ELISA is the most prevalent antibody used for the gluten analysis [6, 14]. RIDASCREEN^® ELISA together with the combination of the cocktail solution has been endorsed as Codex Type 1 Method the Codex Alimentarius as a Type 1 method to determine gluten in different food matrices including corn-based bread, rice-based dough, wheat starches, rice, and corn flour [14, 19]. In 2012, the method was approved as First Action Official Method^®2012.01 [14, 19].

Recently in 2022, a collaborative study has been conducted to demonstrate the extensive applicability of Official Method 2012.01 for the quantitative analysis of gliadin in a wide range of food matrices including soy, starches, pseudo cereals, legumes, spices, juice, nut nougat crème, cream cheese, pesto, meat, vegetarian meat alternative, cookies, dessert, cake, fish, bread, candies, and potatoes. The study result confirms that the method is accurate and suitable to measure gliadin in important gluten-free food matrixes [19].

A1/G12 antibody-based sandwich ELISA is another frequently used ELISA method to detect gluten in the foodstuff [20, 21]. The G12 antibody efficiently measures toxic prolamin accurately in native and partially hydrolyzed cereals [22, 23].

In the present study, we assessed the efficacy of an improved and rapid version of the traditional well-established A1/G12 antibody-based GlutenTox ELISA, i.e. AOAC PTM approved G12/A1 antibody-based 'Glutentox ELISA rapid G12' developed by Hygiena Diagnóstica España S.L., Sevilla, Spain. Though, a limited number of studies compared both the ELISA methods (A1/G12 and R5).[18, 24, 25]. In our study we assessed the performance of the Glutentox ELISA rapid G12 kit using a range of commercially available gluten-free, partially gluten-free products along with spiked samples. R5 ELISA was used as a reference kit and the gluten quantification as well as the gliadin recovery results obtained from the Glutentox ELISA rapid G12 were matched with the R5 ELISA.

Collection of food products

Food products were purchased randomly from different supermarkets in Ancona, Italy. Commercially available food products ‘containing gluten’, ‘naturally gluten-free’, ‘labeled Gluten-free’ and, food products with the label 'may contain traces of gluten' were included in the range of purchased food products.

Gluten estimation ELISA kits

Gluten quantification in food products was assessed through newly developed G12/A1 antibody-based ‘GlutenTox ELISA rapid G12’ (KIT3075, Hygiena Diagnóstica España S.L., Sevilla, Spain), and the results were compared with Ridascreen^® Gliadin (R5) ELISA, R-7001 (R-Biopharm, Darmstadt, Germany) ELISA kits at the Celiac Disease Research Laboratory, Department of Pediatrics, Polytechnic University of Marche, Ancona, Italy. During each ELISA run, standard and samples were added in duplicates into pre-defined ELISA wells. During the ELISA process, the recommended manufacturer's guidelines were strictly followed for each ELISA method. For both methods, food samples quantified with gluten < 20 mg/kg were considered gluten-free. The absorption value of ELISA standards was assured with the quality assurance certificate provided with the respective ELISA kits. Food products with a gluten level higher than 20 mg/kg were re-extracted and re-analyzed second time in duplicates before confirming the results.

Gliadin reference material

For spiking the food samples, the PWG-gliadin standard (protein content 90.7%, gliadin content 87.6%) was purchased from AGF e.V. (Detmold, Germany). Gliadin stock solution was prepared as per the manufacturer's instructions.

ELISA Kits evaluation strategy

The G12/A1 antibody-based GlutenTox rapid G12 ELISA kit was evaluated in two steps. In the first step, the performance of the ELISA kit was inspected by the determination of the threshold value of the gluten in the different food products (gluten containing as well as gluten-free) i.e. <20 mg/kg (above or below). In the second step (spike and recovery), the accuracy of the quantification was assessed by the quantification of gluten in gluten-free food spiked with PWG-gliadin reference material at different gliadin spike levels.

Statistical analysis

Quantitative variables were summarized as the mean ± SD and 95% confidence interval (CI). Paired t-test was applied to compare the means between the study ELISA kits for both the gluten concentration and PWG-gliadin recovery (%). Cohen's kappa test was run to determine the agreement between the gluten detection ELISA methods. The Pearson correlation analysis was performed to analyze the consistency between both the gluten detection ELISA methods. ELISA results < LOQ were excluded from the statistical tests. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) IBM version 23, Chicago (IL), USA.

Determination of gluten content in gluten-free food samples by G12/A1 antibody-based GlutenTox ELISA rapid G12

The Ridascreen R5 ELISA was performed as described in the instruction provided by the

manufacturer. The same procedure was used in previous work [26]. The major steps are

summarized in the following sections.

Sample extraction

Briefly, 0.5 grams (g) of homogenized food samples were measured in a sterile tube, and 5 ml of universal gluten extraction solution (UGES) (patent ES 2 392 412 A1) was added. Tubes were appropriately closed, and a vigorous vortex was provided. In the case of liquid samples, a 0.5 ml food sample was measured and 4.5 ml of UGES was added and vortex for 1–2 minutes. Further, tubes were kept in a water bath at 50^oC for 40 minutes and mixed periodically during incubation. In the case of non-heat-processed/simple matrix composition samples/liquid samples without solids, the samples were incubated at room temperature (20-26^oC) for 40 minutes with mild agitation. At the final step (for all types of samples), tubes were centrifuged at 2500 g for 10 minutes and transferred the supernatant in a fresh sterile tube.

Gluten quantification

Extracted food samples were treated with the provided ready-to-use sample diluent. In each ELISA run, 100 µL of standard, positive control, negative control, internal control, and sample were loaded into the pre-defined well, and the ELISA plate was incubated for 30 minutes at room temperature. An ELISA wash (comprised of five subsequent washes) was provided to the ELISA plate with 300 µL diluted wash buffer and 100 µL of the conjugate antibody (A1-HRP antibody) was added in each well, and another 30 minutes of incubation was provided at room temperature. After another wash, ELISA wells were treated with 100 µL of the substrate solution after another 30 minutes of incubation, the reaction was stopped by adding 100 µL stop solution and ELISA plates were read in ELISA reader (Thermo Scientific multiscanGO) at 450 nm of absorbance).

Determination of gluten content in gluten-free food samples by R5 anbody-based Ridascreen Gliadin R5 ELISA

The Ridascreen R5 ELISA was performed as described in the instruction provided by the manufacturer. The same procedure was used in previous work [26]. The major steps are summarized in the following sections.

Extraction and preparation of samples

Five g of each food sample were homogenized, 0.25 g of homogenized samples were measured and 2.5 ml of recommended cocktail solution (R-7016) (patent WO 02/092633 A1) was added in each tube and heated in a water bath at 50^oC for 40 minutes followed by a treatment of 7.5 ml with freshly prepared 80% ethanol. Tubes were centrifuged (at least 2500 g for 10 minutes), and the supernatant was collected.

Gluten quantification

Extracted samples were diluted at 1:12.5 ratio in provided sample dilution buffer, standard and samples were added in duplicates and the conjugate was added to each well followed by a wash of ELISA plate and incubated for 30 minutes at room temperature. Substrate and chromogen were added, and the ELISA reaction was terminated using stop solution and reading was obtained at the absorbance of 450 nm (thermos scientific multiscanGO).

Both the ELISA methods initially calculated the gliadin concentration in ng/ml. This result was multiplied by the recommended dilution factor obtaining the value of gliadin in corresponding mg/kg. This value was then multiplied by 2 in order to obtain the final level of gluten concentration in food products (gliadin usually represents 50% of the proteins present in gluten) [27].

Major differences between the G12/A1 antibody-based ‘Glutentox ELISA rapid G12’ and R5 antibody-based Ridascreen Gliadin R5 ELISA methods are shown in Table 1.

Table 1

Major differences between the G12/A1 antibody-based ‘Glutentox ELISA rapid G12’ and R5 antibody-based Ridascreen Gliadin R5 ELISA methods
	Glutentox ELISA rapid G12 (KIT-3075)	Ridascreen R5 ELISA (R-7001)
Technical
ELISA kit Brand name	GlutexTox ELISA RAPID G12	Ridascreen R5 ELISA
Validation status	AOAC PTM	AOAC-OMA (2012.01), Codex Alimentarius Method type 1
ELISA type	Sandwich	Sandwich
Coated antibody	G12 antibody (capture antibody) A1-HRP antibody (detection antibody) as conjugate	R5 (monoclonal antibody)
Developed against	α-2-gliadin from 33-mer toxic peptides of the gliadin	ώ-scalin from rye
Target sequence	QLPYPQP (G12) QPQLPY (A1)	QQPFP, QQQFP, LQPFP, QLPFP
Limit of detection (LOD)	0.3 mg/kg gluten	1 mg/kg gluten
Limit of quantification (LOQ)	0.6 mg/kg gluten	5 mg/kg gluten
Gluten extraction
Gluten extraction solution	Universal Gluten Extraction Solution (UGES) (patent ES 2 392 412 A1)	Cocktail solution R-7016 (patent WO 02/092633 A1)
Sample measurement	0.5 g/0.5 ml	0.25 g/0.25 ml
Gluten extraction solution	5 ml	2.5 ml
Incubation (water bath at 50^oC)	40 minutes (conditional)⁺⁺	40 minutes
Ethanol treatment	Not required	7 ml
Shake	Not required	60 minutes
Centrifuge	2500 g for 10 minutes	2500 g for 10 minutes
Total standard duration	50 minutes	110 minutes
ELISA Method
Sample dilution	950 µl dilution x 50 µl sample*	920 µl dilution x 80 µl sample*
Number of gliadin calibrators	5 (1.56 to 50 ng/ml ), ready-to-use	6 (0 to 80 ng/ml ) ready-to-use
Positive control	Yes	NO
Negative control	Yes	NO
Internal control	Yes	NO
ELISA plate wash	300 µl of diluted wash buffer x 5 times	250 µl of diluted wash buffer x 3 times
Conjugate	100 µl (A1-HRP conjugate antibody )	100 µl diluted (HRP based)
Substrate	100 µl	50 µl subtrate + 50 µl chromogen
Stop solution	100 µl	100 µl
First/Second/third incubation	30/30/30 minutes at room temperature (15-25^oC)	30/30/30 minutes at room temperature (15-25^oC)
Overall standard ELISA Duration including gluten extraction	140 minutes (2 hours 20 minutes)	200 minutes (3 hours 20 minutes)
*Suggested sample dilution plan for gluten-free samples; ++ for heat processed samples and/or complex matrix composition, or samples containing polyphenols, tannins or antioxidants.

Spike and recovery test

The PWG-gliadin standard stock solution was prepared by dissolving 1 mg/ml gliadin standard in 60% (v/v) ethanol corresponding to 876 µg/ml gliadin. This stock solution was diluted with the sample dilutions of the respective ELISA kits to prepare different concentrations of gliadin reference material (0,5,10,20,50 and 80 mg/kg) for the spiking experiments. The respective amount of gliadin reference solution was added in the food products during the gluten extraction step. Afterward, the spiked sample extraction and the respective ELISAs were performed as described in the ELISA methodology. The gluten recovery (R) was calculated from the average ELISA concentration (M) and spiked level (S) using the equation R= (M/S) x 100 [28]. Recovery of gliadin standards between the range of 80–120% was considered ideal, and recovery between 50–150% was considered relevant as per the already developed spike and recovery protocol by Abbott et al., 2010 [29]. (Table 1)

Comparison of the ELISA kits for the testing of gluten traces in food products

From December 2022 to May 2023, 47 commercially available food products (‘containing gluten’, ‘naturally gluten-free’, ‘labeled Gluten-free’ and, food products with the label 'may contain traces of gluten' ) were included in the study and clustered into five different food categories, i.e., dairy products (n = 2), legumes (n = 7), meat products (n = 6), cereals (n = 19), and bakery products (n = 13). Of the total food products, 23 were naturally gluten-free, 6 were with ‘gluten-free’ label, 11 were with the label 'may contain gluten traces’ and 7 had gluten as one of the main ingredients. Out of total food samples (n = 47), 29 products were quantified within the accepted gluten threshold (< 20 mg/kg) hence considered gluten-free. Of these 29 gluten-free food, 23 products were detected with less than the limit of quantification (LOQ) by both ELISA kits. However, one product was quantified with < LOQ by G12/A1 ELISA but R5 ELISA detected it within the acceptable level of gluten (5.9 mg/kg) i.e. 5–20 mg/kg. Other 5 food products were quantified within the acceptable level of gluten from both the Kits i.e. <200 mg/kg gluten (Table 2). Remaining 18 food products [‘containing gluten’ (7/7), ‘naturally gluten-free’ (1/23), ‘labeled Gluten-free’ (0/6) and, food products with the label 'may contain traces of gluten' 10/11)] were quantified with a low level of gluten (20–100 mg/kg) by both the kits (Table 2). A good agreement was observed between both the ELISA methods moreover, paired t-test showed no significant difference in the mean gluten concentration between the ELISA kits (p = 0.32). (Table 2)

Table 2

Quantification of gluten (mg/kg) in different food products by G12/A1 antibody-based Glutentox ELISA rapid G12 and R5 antibody-based Ridascreen Gliadin R5 ELISA
Food ID	Food Item	Label	Glutentox ELISA rapid G12 (mg/kg)	Ridascreen Gliadin R5 ELISA (mg/kg)
	Dairy
326	White yogurt	Naturally gluten-free	< LOQ	< LOQ
439	Flavoured Yogurt	Gluten-free	< LOQ	< LOQ
	Legumes
420*	Lentils	Naturally gluten-free	< LOQ	< LOQ
445	Mix of legumes	May contain gluten traces	34.2	26.9
341*	Chickpeas	Naturally gluten-free	< LOQ	< LOQ
443	Chickpea flour	May contain gluten traces	49.0	32.6
448	Green peas canned	Naturally gluten-free	3.1	16
464	Lentil mix	May contain gluten traces	41.5	38.3
469	Red lentils	Naturally gluten-free	1.32	8.25
	Meat
343	Lamb	Naturally gluten-free	< LOQ	< LOQ
351	Turkey	Naturally gluten-free	< LOQ	< LOQ
373	Beef	Naturally gluten-free	< LOQ	< LOQ
390	Pork	Naturally gluten-free	< LOQ	5.9
438*	Chicken	Naturally gluten-free	< LOQ	< LOQ
325	Fish	Naturally gluten-free	< LOQ	< LOQ
	Cereals
305*	Rice flour	Naturally gluten-free	< LOQ	< LOQ
349	Quinoa mixed seeds	Naturally gluten-free	< LOQ	< LOQ
364	Kidney bean	Naturally gluten-free	< LOQ	< LOQ
376	Rice pasta	Naturally gluten-free	< LOQ	< LOQ
397*	Corn pasta	Naturally gluten-free	< LOQ	< LOQ
399	Italian rice	Naturally gluten-free	< LOQ	< LOQ
402	Buckwheat	Naturally gluten-free	< LOQ	< LOQ
417	Poppy seeds	Naturally gluten-free	< LOQ	< LOQ
440	Corn flakes	Naturally gluten-free	< LOQ	< LOQ
441	Millet	May contain gluten traces	< LOQ	< LOQ
444	Oat bran	May contain gluten traces	36.9	45.8
442	Vegetable soup	Gluten-free	11.8	13.6
457	Corn semolina	Contains soft wheat	40	49.1
458	Oat flour	Naturally gluten-free	1.7	5.5
461	Corn cereal	May contain gluten traces	34.9	54.2
463	Whole chickpeas	May contain gluten traces	42.4	41.5
465	Buckwheat	May contain gluten traces	36	39.9
467	Amaranth	May contain gluten traces	48.8	47.3
472	Quinova seeds	Naturally gluten-free	7.64	2.4
	Bakery
446*	Crackers	Gluten-free	< LOQ	< LOQ
403	Cacao powder	Gluten-free	< LOQ	< LOQ
406	Biscuits	Gluten-free	< LOQ	< LOQ
416	Bread	Gluten-free	< LOQ	< LOQ
447	Barley wafer	Contains barley	92.9	80.7
449	Cereal bar (Braley)	Contains barley	79.83	80.25
450	Cereal bar (Wheat)	Contains wheat	74.4	74.5
452	Puffed pastries	Contains wheat	63.7	79.7
454	Sesame sticks	Contains soft wheat	56.9	73
460	Potato chips	May contain gluten traces	50.5	58.5
462	Barley flakes	Contains barley	39.8	33.9
466	Salted peanuts	May contain gluten traces	54	48.9
455	Coconut powder	Naturally gluten-free	35.9	31.8
*Food products also used for spike and recovery test

Accuracy of the quantification of the G12/A1 antibody-based GlutenTox rapid G12 ELISA and its coparison with R5 antibody-based Ridascreen R5 ELISA: spike and recovery test

To evaluate the matrix effect of the ELISA kits, a spike and recovery test was performed using PWG-gliadin reference material. For this purpose, six food products found gluten-free by both ELISA kits were chosen from the original samples list (Table 2). These products were chicken (Sample ID 438 ), chickpeas (sample ID 341), lentil (Sample ID 420), corn pasta (sample ID 397), gluten-free crackers (sample ID 446), and rice flour (sample ID 305). They were chosen among items characterized by a difficult gluten extraction process due to high-fat content (cracker), extruded food products (pasta), and legumes (Wieser et al. 2008).

Recovery results showed an overall mean recovery for GlutenTox rapid G12, and R5 ELISA of 97.4 ± 19.4 (95% CI: 91–104; range: 74–141 mg/kg), and 99.4 ± 18.2 (95% CL: 92–105; range: 66–141 mg/kg), respectively. Figure 1 shows a correlation in the gluten concentration and recovery obtained from GlutenTox G12 rapid ELISA and Ridascreen R5 ELISA. The recovery rate of G12/A1 GlutenTox rapid G12 kit and Ridascreen R5 ELISA were similar, and the average mean of recovery for the PWG-gliadin standard was in the range of 50–150% for both the ELISA methods (Fig. 2). A similar pattern was achieved on the spiked food matrices mean recovery of PWG-gliadin standard (Table 3). There was a consistency found in the mean gluten concentration mean gluten recovery in the individual kits. However, paired t-test between both the ELISA kits showed no significant differences in gluten concentration and recovery means (p = 0.48). Additionally, Cohen's kappa test showed good strength of agreement between the two methods in terms of recovery, k = 0.66 (95% CI, 0.39 to 0.93), p < 0.001 (individual categories were < 50%, 50–100%, > 100%). (Table 3 ) (Fig. 1 and Fig. 2 )

Table 3

Recovery (%) of PWG-gliadin reference material by both the ELISA methods in spiked food matrices at different spike levels (0, 5, 10, 20, 50, and 80 mg/kg)
Food items	0 mg/kg (unspiked)	PWG-gliadin 5 mg/kg (S) recovery (R) (Raw data; M)	PWG-gliadin 10 mg/kg (S) recovery (R) (Raw data; M)	PWG-gliadin 20 mg/kg (S) recovery (R) (Raw data; M)	PWG-gliadin 50 mg/kg (S) recovery (R) (Raw data; M)	PWG-gliadin 80 mg/kg (S) recovery (R) (Raw data; M)
GlutenTox rapid G12 ELISA
Chicken	< LOQ	125 (6.2)	110 (11.0)	85 (17.1)	75 (37.5)	81 (65.45)
Chickpea flour	< LOQ	134 (6.7)	102 (10.2)	85 (17.1)	84 (42.2)	82 (65.9)
Lentil	< LOQ	122 (6.1)	102 (10)	99 (19.8)	84 (42.2)	88 (70.7)
Corn pasta	< LOQ	141 (7.0)	115 (11.5)	75 (15.0)	81 (40.9)	78 (62.9)
Cracker	< LOQ	130 (6.5)	103 (10.3)	102 (20.4)	82 (41.3)	84 (67.4)
Rice flour	< LOQ	118.4 (5.9)	107 (10.7)	86 (17.3)	84 (42.0)	74 (59.9)
Mean ± SD	N/A	129 ± 8.5*	106 ± 5.2*	89 ± 10*	82 ± 3.6*	81 ± 4.6*
Ridascreen Gliadin R5 ELISA
Chicken	< LOQ	120 (6.0)	105 (10.5)	102 (20.5)	73 (36.6)	87 (69.8)
Chickpea flour	< LOQ	129 (6.5)	114 (11.4)	91 (18.3)	69 (48.0)	92 (74.1)
Lentil	< LOQ	107 (5.3)	95 (9.5)	81 (16.3)	91 (45.8)	95 (76.6)
Corn pasta	< LOQ	126 (6.3)	113 (11.3)	77 (15.5)	81 (40.7)	86 (69.3)
Cracker	< LOQ	141 (7.0)	109 (10.9)	92 (18.4)	118 (59.3)	66 (53.4)
Rice flour	< LOQ	128 (6.4)	81 (8.1)	93 (18.6)	87 (43.7)	93 (75.1)
Mean ± SD	N/A	125 ± 11*	103 ± 12*	90 ± 9*	91 ± 16*	87 ± 10*
*Indicates mean ± SD (standard deviation), LOQ is the limit of quantification.
Gliadin recovery values expressed in %., PWG: prolamin working group, N/A: not applicable

Gluten is a pervasive protein used in diverse commercial food and sometimes even non-food products (e.g., toothpaste, hair shampoo, and cosmetics) [6, 27]. Unintentional ingestion of gluten traces may damage the intestinal integrity of CD patients [7, 31]. Hence, it is essential to know the exact amount of gluten in a food product to provide safe gluten-free products to each CD patient [13].

Several antibodies have been developed for gluten detection in food products. However, among them, R5 antibody-based ELISA method is a most prevalent method for gluten detection in food stuff [14]. On the other hand, G12 antibody-based ELISA method is another frequently used method for the same purpose [20, 21]. The R5 monoclonal antibody (mAb) is raised against the omega-Secalin from rye, strongly recognizes the most toxic fragments of gliadin i.e. QQPFP, QQQFP, LQPFP, and QLPFP sequences [32]. G12 and A1 are highly sensitive mAb antibodies developed against the α2-gliadin 33-mer toxic peptide of the gliadin [22]. The G12 mAb exhibits an affinity for the toxic peptide eight times higher than A1 mAb. The traditional Biomedal G12 ELISA method (KT-5139, Biomedal diagnostics, Spain) was based on A1 and G12 antibodies where A1 antibody (embedded on the ELISA wells) was used as the capture antibody, and G12 antibody (conjugate) was used as the detection antibody [22]. These antibodies are claimed to have a broader specificity for the prolamins that are more toxic for CD patients. Hence, it is highly suitable for the detection of gliadin from the food. The G12 antibody proved to be efficient in measuring prolamin concentrations in both native and partially hydrolyzed cereals [22, 23].

The G12/A1 antibody-based GlutenTox rapid G12 ELISA Kit has AOAC PTM approval with PTM certificate No. #042301. In this kit, the G12 mAb (capture antibody) is coated on the ELISA well, and treatment of A1-HRP antibody (detection antibody) is provided as conjugate, which is precisely the reverse of the traditional GlutenTox G12 test. The G12 antibody is restricted to the detection of the QPQLPY sequence of 33-mer toxic gliadin fragment while A1 has a broader range of epitope recognition, and apart from the QLPYPQP sequence, it also recognizes for two more sequences (QQPFPQP and QLPFPQP) [22]. Therefore, the reversal of the location of antibodies (G12 and A1) is an improved fit with a polynomial function.

As for the ability to detect the threshold quantity of the gluten (> 20 mg/kg) we found no significant difference in the mean gluten concentration between the tested ELISA kits (p = 0.32) (Table 2). This result is similar to the study conducted by Hochegger R. et al.,2015 where the authors compared R5 antibody-based ELISA with A1/G12 antibody-based conventional GlutenTox G12 ELISA kits in various food products (flours, bakery, and soy products) and found similar results using either R5 or conventional G12 method [33].

The PWG-gliadin recovery results showed no significant difference in the recovery percentage mean (p = 0.48) and found a strong agreement between both the ELISA kits as analyzed by the kappa test (K = 0.66). Recovery of gliadin standard constantly remained in the acceptable range ( 50–150% ) and yielded consistent results by both the kits (Fig. 2) [29]. Moreover, raw data obtained by G12/A1 antibody-based ‘GlutenTox rapid G12’ ELISA was accurate against the spike levels (Table 3). However, in a study Bruins Slot et al.,2015 evaluated the performance of 14 different gluten ELISA kits (including Ridascreen R5 and Biomedal GlutenTox G12 ELISA) and concluded that no gluten kit accurately quantifies gluten in different matrices. Nonetheless, in the same work, the authors showed an acceptable recovery rate by Ridascreen R5 and conventional GlutenTox G12 ELISA [34].

For gluten extraction, the R5 ELISA uses a patented (WO 02/092633 A1) cocktail solution (R7006) containing hazardous chemicals such as ‘β-mercaptoethanol’ as reducing agent and another potent corrosive denaturant (Guanidinium chloride) that require attention to the user performing the method.[35] However, it is a widely accepted gluten extraction method [36, 37]. Allred and Ritter 2010, compared the cocktail and ethanol-based gluten extraction methods and reported that reducing agent-containing cocktail solution provides a better gluten extraction result [38]. Some other studies also reported an extensive difference in the gluten amount obtained from the cocktail solution with reducing agents in comparison to the ethanol-based extraction solution [38, 39].

In contrast to Ridascreen R5 ELISA, G12/A1 antibody-based GlutenTox G12 rapid ELISA uses a patented (ES2 393 412 A1) UGES that contains reducing agents, disaggregating and solubilizing reagents. UGES is based on hydroalcoholic solution (less than 40%) and arginine that promotes gluten solubility. In a recent study Segura et. al demonstrated that the UGSE is effective even for heat-treated and hydrolyzed foods and also compatible with various immunological methods and has no toxicity to the user and the environment [40].

In our study, we included 47 food products for the gluten quantification, and for the spike and recovery test, we selected 6 out of 47 food products (that were detected gluten-free by both GlutenTox rapid G12 and Ridascreen R5 gluten ELISA) for the recovery test. These food matrices make the recovery process complicated. Additionally, we used PWG-gliadin standard reference material for the spike and recovery test at 6 different spike levels (i.e. 0, 5, 10, 20, 50, and 80 mg/kg) in duplicates. These steps convinced us of our findings. However, we recommend further validation of the G12/A1 antibody-based ‘GlutenTox rapid G12’ ELISA with an increased number of food products.

G12/A1 antibody-based GlutenTox rapid G12 test is an appropriate choice for the detection of gluten in food products and performs accurately as the current reference method, i.e., R5 ELISA. The advantages of this new method are: (a) it is a faster gluten detection method; (b) it does not use potentially hazardous chemicals. The G12/A1 antibody-based ‘GlutenTox ELISA rapid G12’ method could be a fair alternative gluten ELISA test to the Ridacreen R5 ELISA test. However, more studies with further investigation (e.g. more in-house validation) and an increased number of food products are required to support the study findings.

Acknowledgments: The authors acknowledge the support of Dr. Carlos Galera (Hygiena Diagnóstica España S.L., Sevilla) for providing the Glutentox ELISA rapid G12 test kit and for the technical support.

Funding statement: This research received no external funding.

Data availability: Study data has been given within the manuscript.

Conflicts of interest: Authors declare no conflicts of interest.

Megiorni F, Mora B, Bonamico M, Barbato M, Nenna R, Maiella G et al (2009) HLA-DQ and risk gradient for celiac disease. Hum Immunol 70:55–59. https://doi.org/10.1016/j.humimm.2008.10.018
Gatti S, Lionetti E, Balanzoni L, Verma AK, Galeazzi T, Gesuita R et al Increased Prevalence of Celiac Disease in School-age Children in Italy. Clin Gastroenterol Hepatol 2019:S1542356519306512. https://doi.org/10.1016/j.cgh.2019.06.013
Ciacci C, Ciclitira P, Hadjivassiliou M, Kaukinen K, Ludvigsson JF, McGough N et al (2015) The gluten-free diet and its current application in coeliac disease and dermatitis herpetiformis. United Eur Gastroenterol J 3:121–135. https://doi.org/10.1177/2050640614559263
Hall NJ, Rubin G, Charnock A (2009) Systematic review: adherence to a gluten-free diet in adult patients with coeliac disease. Aliment Pharmacol Ther 30:315–330. https://doi.org/10.1111/j.1365-2036.2009.04053.x
Lionetti E, Catassi C (2011) New clues in celiac disease epidemiology, pathogenesis, clinical manifestations, and treatment. Int Rev Immunol 30:219–231. https://doi.org/10.3109/08830185.2011.602443
Verma A, Gatti S, Galeazzi T, Monachesi C, Padella L, Baldo G et al (2017) Gluten Contamination in Naturally or Labeled Gluten-Free Products Marketed in Italy. Nutrients 9:115. https://doi.org/10.3390/nu9020115
Catassi C, Fabiani E, Iacono G, D’Agate C, Francavilla R, Biagi F et al (2007) A prospective, double-blind, placebo-controlled trial to establish a safe gluten threshold for patients with celiac disease. Am J Clin Nutr 85:160–166. https://doi.org/10.1093/ajcn/85.1.160
Codex Alimentarius Commission (2008) Codex Alimentarius CommissionFoods for Special Dietary Use for Persons Intolerant to Gluten Codex STAN 118–1979 Codex Alimentarius Commission: Rome, Italy,2008
European Commission COMMISSIONREGULATION, No (2009) 41/2009 of 20 January 2009 concerning the composition and labelling of foodstuffs suitable for people intolerant to gluten, http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:016:0003:0005:EN:PDF. 2009
Food US, Administration D (2013) Food labeling; gluten-free labeling of foods, final rule. Fed. Regist. 78:47154–47179. Available at: https://www.federalregister.gov/articles/2013/08/05/2013-18813/ food-labeling-gluten-free-labeling-of-foods. 2013
Lee HJ, Anderson Z, Ryu D (2014) Gluten contamination in foods labeled as gluten free in the United States. J Food Prot 77:1830–1833. https://doi.org/10.4315/0362-028X.JFP-14-149
Koerner TB, Cléroux C, Poirier C, Cantin I, Alimkulov A, Elamparo H (2011) Gluten contamination in the Canadian commercial oat supply. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 28:705–710. https://doi.org/10.1080/19440049.2011.579626
Osorio, Mejías (2019) Rustgi. Gluten Detection Methods and their Critical Role in Assuring Safe Diets for Celiac Patients. Nutrients 11:2920. https://doi.org/10.3390/nu11122920
Scherf KA, Catassi C, Chirdo FG, Ciclitira PJ, Feighery CF, Gianfrani C et al (2021) Statement of the Prolamin Working Group on the Determination of Gluten in Fermented Foods Containing Partially Hydrolyzed Gluten. Front Nutr 7:626712. https://doi.org/10.3389/fnut.2020.626712
Panda R, Garber EAE (2019) Detection and Quantitation of Gluten in Fermented-Hydrolyzed Foods by Antibody-Based Methods: Challenges, Progress, and a Potential Path Forward. Front Nutr 6:97. https://doi.org/10.3389/fnut.2019.00097
Diaz-Amigo C, Popping B (2013) Accuracy of ELISA Detection Methods for Gluten and Reference Materials: A Realistic Assessment. J Agric Food Chem 61:5681–5688. https://doi.org/10.1021/jf3046736
Sharma GM, Khuda SE, Pereira M, Slate A, Jackson LS, Pardo C et al (2013) Development of an Incurred Cornbread Model for Gluten Detection by Immunoassays. J Agric Food Chem 61:12146–12154. https://doi.org/10.1021/jf404072x
Hochegger R, Mayer W, Prochaska M (2015) Comparison of R5 and G12 Antibody-Based ELISA Used for the Determination of the Gluten Content in Official Food Samples. Foods 4:654–664. https://doi.org/10.3390/foods4040654
Lacorn M, Dubois T, Weiss T, Zimmermann L, Schinabeck T-M, Loos-Theisen S et al (2022) Determination of Gliadin as a Measure of Gluten in Food by R5 Sandwich ELISA RIDASCREEN® Gliadin Matrix Extension: Collaborative Study 2012.01. J AOAC Int 105:442–455. https://doi.org/10.1093/jaoacint/qsab148
Escarnot E, Gofflot S, Sinnaeve G, Dubois B, Bertin P, Mingeot D (2018) Reactivity of gluten proteins from spelt and bread wheat accessions towards A1 and G12 antibodies in the framework of celiac disease. Food Chem 268:522–532. https://doi.org/10.1016/j.foodchem.2018.06.094
Silvester JA, Comino I, Rigaux LN, Segura V, Green KH, Cebolla A et al (2020) Exposure sources, amounts and time course of gluten ingestion and excretion in patients with coeliac disease on a gluten-free diet. Aliment Pharmacol Ther 52:1469–1479. https://doi.org/10.1111/apt.16075
Morón B, Cebolla A, Manyani H, Alvarez-Maqueda M, Megías M, Thomas MDC et al (2008) Sensitive detection of cereal fractions that are toxic to celiac disease patients by using monoclonal antibodies to a main immunogenic wheat peptide. Am J Clin Nutr 87:405–414. https://doi.org/10.1093/ajcn/87.2.405
Morón B, Bethune MT, Comino I, Manyani H, Ferragud M, López MC et al (2008) Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide. PLoS ONE 3:e2294. https://doi.org/10.1371/journal.pone.0002294
Bruins Slot ID, Bremer MGEG, van der Fels-Klerx I, Hamer RJ (2015) Evaluating the Performance of Gluten ELISA Test Kits: The Numbers Do Not Tell the Tale. Cereal Chem J 92:513–521. https://doi.org/10.1094/CCHEM-07-14-0166-R
Yu JM, Lee JH, Park J-D, Choi Y-S, Sung J-M, Jang HW (2021) Analyzing Gluten Content in Various Food Products Using Different Types of ELISA Test Kits. Foods 10:108. https://doi.org/10.3390/foods10010108
Verma AK Identification of a new biomarker for celiac disease and other gluten-related disorders. Università Politecnica delle Marche, n.d
Verma AK, Lionetti E, Gatti S, Franceschini E, Catassi GN, Catassi C (2019) Contribution of Oral Hygiene and Cosmetics on Contamination of Gluten-free Diet: Do Celiac Customers Need to Worry About? J Pediatr Gastroenterol Nutr 68:26–29. https://doi.org/10.1097/MPG.0000000000002129
Sharma GM, Rallabhandi P, Williams KM, Herrmann M, Sadrieh N Gluten Quantitation in Cosmetic Products by Enzyme-Linked Immunosorbent Assay 2016. https://doi.org/info:doi/10.5740/jaoacint.16-0037
Abbott M, Hayward S, Ross W, Godefroy SB, Ulberth F, Van Hengel AJ et al (2010) Validation procedures for quantitative food allergen ELISA methods: community guidance and best practices. J AOAC Int 93:442–450
Wieser H (2008) Detection of gluten. Pages 47–80 in: Gluten-Free Cereal Products and Beverages. E. K. Arendt and F. Dal Bello, eds. Elsevier: London, U.K. Elsevier; n.d
Syage JA, Kelly CP, Dickason MA, Ramirez AC, Leon F, Dominguez R et al (2018) Determination of gluten consumption in celiac disease patients on a gluten-free diet. Am J Clin Nutr 107:201–207. https://doi.org/10.1093/ajcn/nqx049
Osman AA, Uhlig HH, Valdes I, Amin M, Méndez E, Mothes T (2001) A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins. Eur J Gastroenterol Hepatol 13:1189–1193
Hochegger R, Mayer W, Prochaska M (2015) Comparison of R5 and G12 Antibody-Based ELISA Used for the Determination of the Gluten Content in Official Food Samples. Foods 4:654–664. https://doi.org/10.3390/foods4040654
Bruins Slot ID, Bremer MGEG, van der Fels-Klerx I, Hamer RJ (2015) Evaluating the Performance of Gluten ELISA Test Kits: The Numbers Do Not Tell the Tale. Cereal Chem J 92:513–521. https://doi.org/10.1094/CCHEM-07-14-0166-R
Melini F, Melini V (2018) Immunological Methods in Gluten Risk Analysis: A Snapshot. Safety 4:56. https://doi.org/10.3390/safety4040056
Mena MC, Lombardía M, Hernando A, Méndez E, Albar JP (2012) Comprehensive analysis of gluten in processed foods using a new extraction method and a competitive ELISA based on the R5 antibody. Talanta 91:33–40. https://doi.org/10.1016/j.talanta.2011.12.073
Bermudo Redondo MC, Griffin PB, Garzon Ransanz M, Ellis HJ, Ciclitira PJ, O’Sullivan CK (2005) Monoclonal antibody-based competitive assay for the sensitive detection of coeliac disease toxic prolamins. Anal Chim Acta 551:105–114. https://doi.org/10.1016/j.aca.2005.07.023
Allred LK, Ritter BW (2010) Recognition of gliadin and glutenin fractions in four commercial gluten assays. J AOAC Int 93:190–196
Sharma GM (2012) Immunoreactivity and Detection of Wheat Proteins by Commercial ELISA Kits. J AOAC Int 95:364–371. https://doi.org/10.5740/jaoacint.SGE_Sharma
Segura V, Díaz J, Ruiz-Carnicer Á, Muñoz-Suano A, Carrillo-Carrión C, Sousa C et al (2021) Rapid, Effective, and Versatile Extraction of Gluten in Food with Application on Different Immunological Methods. Foods 10:652. https://doi.org/10.3390/foods10030652

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Performance assessment of a new G12/A1 antibody-based rapid ELISA using commercially available and gluten-spiked food samples

Status:

Version 1

Abstract

Background

Aims

Methods

Results

Conclusions

Figures

Introduction

Methods

Collection of food products

Gluten estimation ELISA kits

Gliadin reference material

ELISA Kits evaluation strategy

Statistical analysis

Determination of gluten content in gluten-free food samples by G12/A1 antibody-based GlutenTox ELISA rapid G12

Sample extraction

Gluten quantification

Extraction and preparation of samples

Gluten quantification

Spike and recovery test

Results

Comparison of the ELISA kits for the testing of gluten traces in food products

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1