Validation of the GlutenTox® ELISA Rapid G12 Test Kit for Determination of Gluten in Select Non-Heat-Processed Matrixes and Heat-Processed Matrixes: AOAC Performance Tested MethodSM 042301

Abstract Background The GlutenTox® ELISA Rapid G12 test kit is a quantitative method designed for the determination of the immunotoxic fraction of gluten in food samples. Objective To obtain AOAC Performance-Tested MethodsSM certification for the method for the detection and quantification of gluten from wheat, barley, and rye flours in select foods (non-heat-processed) and incurred (heat-processed) matrixes. Methods The method was evaluated following the Guidelines for Validation of Quantitative Gluten Methods, with Specific Examples for ELISA Assays. The validation study was conducted at Hygiena Diagnóstica España using five food matrixes (soy flour, corn bread, seasoning mix, rolled oats, and evaporated milk) artificially contaminated with gluten from wheat, barley, or rye flour at different concentrations: 0, 5, 10, and 20 mg/kg. For each matrix and gluten contamination level, five or six individually extracted test portions were analyzed. A second bread matrix was prepared by baking a gluten-free bread mix spiked at 0, 20, and 30 mg/kg gluten from wheat, barley, or rye flour for incurred matrix testing. Ten individually extracted test portions were tested for each incurred bread and contamination level of gluten. Results The method met the AOAC performance requirements for detection and quantification of wheat gluten in the selected food matrixes, incurred bread sample, and spike levels of wheat gluten, showing an acceptable recovery. When tested with barley and rye flours, most of the results showed acceptable recoveries or a slight overestimation, depending on the matrix and gluten concentration. Method developer and independent laboratory results were comparable. Conclusions The validation study demonstrated that the test kit is a reliable, accurate, quick, and easy-to-use method for the detection and quantification of gluten concentration in food and incurred matrixes from wheat, barley, and rye flours. Highlights Most reagents provided in the kit are at ready-to-use concentrations.


General Information
Gluten is a protein fraction from wheat, rye, barley, oats, or their crossbred varieties and derivatives thereof, to which some persons are intolerant and which is insoluble in water and 0.5 M NaCl (1).
An inappropriate response of the immune system to gluten causes celiac disease, a disorder that damages the small intestine, causing the atrophy of the intestinal villi, which interferes with the absorption of nutrients such as proteins, lipids, carbohydrates, mineral salts, and vitamins.This disease leads to diarrhea, vitamin and mineral deficiencies, anemia, and thin bones (osteoporosis) and affects people of all ages.
Currently, the only treatment for celiac disease sufferers is a strict, lifelong gluten-free diet, which presents great difficulties because gluten, in addition to being present in many foods, may also be found in food additives and preservatives.

Principle
The GlutenTox V R ELISA Rapid G12 method is a sandwich ELISA assay that can be used to detect and quantify gluten in food samples.
To solubilize the gluten present in the sample's matrix, the extraction solution (UGES) (2) provided in the kit is added to the food sample.
After the extraction, the sample's extract is added to a multiwell plate coated with a monoclonal anti-gliadin antibody (G12) that specifically recognizes the most immunogenic fraction of gluten.After the washing steps, the addition of a second monoclonal anti-gliadin antibody conjugated to HRP (A1-HRP) and the substrate solution (TMB) will allow for the measurement of the signal (color change).GlutenTox ELISA Rapid G12 is a direct method.The higher the concentration of gluten present in the sample, the more intense the signal will be.Conditions where test results or measurement results are obtained with the same method, on identical test/measurement items in the same test or measurement facility, under different operating conditions (4).(d) Linearity.-Linearity of the method extending beyond the set of standards or calibrators supplied with the kit.Direct and unambiguous relationship between measurement response and concentration.(e) Calibrant.-Amaterial used for calibration of a measurement procedure.(f) Selectivity.-Ability of the method to detect analyte without interference from matrix or other components of similar behavior.(g) Relative recovery.-Recovery is the ratio of the mean candidate method result to the true value or reference method value, expressed as a percentage, [mean cand /known spike] Â 100 or [mean cand /mean ref ] Â 100.(h) Bias.-Bias is the difference between the candidate method mean result and the true value or reference method value, mean cand-known spike or mean cand-mean ref .

Scope of Method
Bias is the total systematic error as contrasted to random error.There may be one or more systematic error components contributing to the bias.The lowest concentration or mass of analyte in a test sample that can be distinguished from a true blank sample at a specified probability level, where X 0 is the mean concentration value of the blank replicates, m is the slope, and s b is the intercept (3).(l) LOQ.-Mean þ 10s r , or 3 Â LOD (if linear regression model is used).
The lowest level of analyte in a test sample that can be reasonably quantified at a specified level of precision (3).

Materials and Methods
Test Kit Information

Safety Precautions
The GlutenTox ELISA Rapid G12 contains material that may cause skin or eye irritation.Wear safety glasses with side shields that have been approved under the appropriate government standards.If reagents come in contact with eyes, remove contacts and flush with water for 15 min.Wear gloves when handling materials to avoid contact with skin.Flush skin with plenty of water if accidental contact occurs.Certain chemicals in the kit should not be released into the environment.

General Preparation
(a) Carefully read the user manual before performing the assay.

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(b) It is recommended that the instructions described in the manual be followed exactly as described.(c) This kit is designed for professional use only.(d) Do not mix components from various kits or use reagents or solutions other than those supplied.(e) Allow all reagents, except GlutenTox A1-HRP conjugated antibody, to reach room temperature (15-25 C/59-77 F) prior to testing.(f) After removing multi-well test strips, reseal the aluminum bag immediately.(g) The wash solution is supplied as a 10Â concentrate, which must be diluted 1:10 in distilled water prior to use.(h) The diluted wash solution remains stable for two weeks at 2-8 C (36-46 F). (i) Each sample material should be analyzed at least in duplicate.

Sample Preparation
Extraction of solid and semisolid samples: prepare dilutions according to Table 1.(e) Add 100 mL of each standard, positive control, negative control, internal control, and sample dilution to separate wells in duplicate.Cover the wells and incubate at room temperature (15-25 C/59-77 F) for 30 min.(f) Eliminate the well contents by inverting the plate.Add 300 mL of diluted wash solution to all wells and incubate for 3 s.Repeat this sequence four more times for a total of five washes.Perform the washes in the same order used to load the wells in the previous step.After the last wash, invert the plate and tap it on an absorbent material such as a clean paper towel to eliminate any remaining liquid.An automatic washer is recommended for a higher reproducibility of the results.(g) Add 100 mL of the GlutenTox A1-HRP conjugated antibody to each well.Cover the wells and incubate at room temperature (15-25 C/59-77 F) for 30 min.(h) Wash the plate five times with 300 mL of diluted wash solution per well as indicated in the previous step.(i) Add 100 mL of substrate solution to each well.Cover the wells and incubate at room temperature (15-25 C/59-77 F) for 30 min in the dark.(j) Add 100 mL of stop solution to each well.Follow the same order used when adding the substrate solution in the previous step.(k) Using an ELISA microplate reader with a 450 nm filter, read the absorbance (OD) of each well as soon as possible, within 30 min of the addition of stop solution.The AOAC OMA 2012.01 method (5) was used to prescreen natural contamination of all matrixes and to verify homogeneity on every food matrix high-level concentration stock.

Reference Materials
Materials used for contamination (wheat, barley, and rye flours) were procured from a retail source and were independently characterized for total protein content.
Wheat flour: Gallo (Carrefour Supermarket, Camas, Sevilla, Spain); Batch L71 018 11 Rye flour: Barry Farm (The Barry Farm, Wapakoneta, OH); Batch 90010 Barley flour (whole grain): Marhaba (Kalustyan's, NY) (a) Wheat flour.-Thewheat flour was tested by the Kjeldahl nitrogen method, obtaining 9.13% protein (9.13 g of protein in 100 g of sample).This value was multiplied by 0.80 (the conversion factor of wheat), resulting in 7.3% gluten (7.3 g of gluten protein in 100 g of sample).This percent value was then multiplied by 10 000 to estimate the mg/kg value at 73 000.This is equivalent to 73 mg of gluten per gram of flour.(b) Barley flour.-Thebarley flour was tested by the Kjeldahl nitrogen method, obtaining 9.94% protein (9.94 g of protein in 100 g of sample).This value was multiplied by 0.75 (the conversion factor of barley), resulting in 7.46% gluten (7.46 g of gluten protein in 100 g of sample).This percent value was then multiplied by 10 000 to estimate the mg/kg value at 74 600.
This is equivalent to 74.6 mg of gluten per gram of flour.(c) Rye flour.-Therye flour was tested by the Kjeldahl nitrogen method, obtaining 8.46% protein (8.46 g of protein in 100 g of sample).This value was multiplied by 0.48 (the conversion factor of rye), resulting in 4.06% gluten (4.06 g of gluten protein in 100 g of sample).This percent value was then multiplied by 10 000 to estimate the mg/kg value at 40 600.This is equivalent to 40.6 mg of gluten per gram of flour.(d) Preparation of high-level gluten source flour concentration stocks.-A75-200 mg/kg (ppm) stock mixture was created by blending the gluten source flour into the matrix material (total weight of 500 g).This mixture was very thoroughly blended to ensure homogeneity.

Preparation of High-Level Wheat Flour Concentration Stocks for Dry Matrix Spiking and Test Portions
A high-level concentration stock of gluten from wheat flour (75-200 mg/kg gluten) was prepared in each food matrix.All matrixes were prescreened using the AOAC OMA 2012.01 method (5) to detect natural contamination prior to the study startup.
Matrixes for spiking were prepared by chopping, grinding, and mixing until they were finely ground and uniform.
From the bulk matrix, 500 g of each gluten-free food matrix was weighed on an analytical balance and added into the bowl mixer (Cecotec Mixer).The spiking reference material needed (from wheat flour) was weighed and slowly added to the glutenfree food matrix in the bowl mixer mixing by hand, thoroughly and constantly, until the mixture appeared fully incorporated.Then, the content of the bowl was first mixed in the mixer at maximum power (1000 w) for 5 min and, subsequently, mixed manually with a spoon for 5 min.This mixing process was repeated four times to achieve a homogeneous distribution of the contaminant within the food matrix.About 20 g of the mixture was aliquoted in a 125 mL polypropylene flat-bottom tube (the rest of the mixture was stored in a self-closing plastic bag in a dry place).
Homogeneity testing on the high concentration stocks was performed in every food matrix stock by analyzing 10 test portions randomly chosen using the AOAC OMA 2012.01 method (5).

Preparation of Dry Test Portions
The high-level wheat flour concentration stocks were diluted, using a stepwise dilution scheme, to prepare gluten-contaminated matrix batches for each level being tested (0, 5, 10, and 20 ppm of gluten from wheat flour).Each contaminated matrix batch went through the same mixing process (as the high concentration stocks) four times.
A portion of 500 g of the 20 mg/kg gluten concentration was prepared for each gluten-free matrix using the finely ground and uniform matrix with the corresponding amount of high-level wheat flour concentration stock.
The 10 mg/kg gluten concentration was prepared for each gluten-free matrix mixing 250 g of finely ground and uniform matrix with 250 g of the corresponding matrix contaminated at 20 mg/kg of gluten.
The 5 mg/kg gluten concentration was prepared for each gluten-free matrix mixing 250 g of finely ground and uniform matrix with 250 g of the corresponding matrix contaminated at 10 mg/kg of gluten.Six individual 0.5 g test portions of every matrix at each concentration of gluten (0, 5, 10, and 20 mg/kg of gluten from wheat flour) were portioned out into 50 mL polypropylene flat-bottom test tubes.Once prepared, the test portions were held at room temperature in a dry place until use.

Preparation of High-Level Wheat Flour Concentration Stocks for Moist Matrix Spiking and Test Portions
A high concentration stock of gluten from wheat flour (128.5 mg/kg gluten) was prepared for the evaporated milk matrix.
This matrix was prescreened using the AOAC OMA 2012.01 method (5) to detect natural contamination prior to the study startup.
The evaporated milk matrix for spiking was prepared by mixing until it was uniform.
Using the candidate method extraction buffer, 90 mL of the high concentration suspension stock of gluten (4000 mg/kg gluten) from wheat flour (3.755 g) was prepared.This suspension was used to spike into the gluten-free evaporated milk matrix.Thus, 15 mL of the high concentration suspension stock of gluten was slowly added to 485 mL of the gluten-free evaporated milk matrix in a bottle, mixing thoroughly and constantly with a magnetic stirrer for 30 min until the mixture (128.5 mg/ kg gluten) appeared fully incorporated.By this mixing process, a homogeneous distribution of the contaminant within the food matrix was achieved.The mixture was stored refrigerated at 4 C. Homogeneity testing on the high concentration stock was performed in the evaporated milk matrix stock by analyzing 10 test portions randomly chosen using the AOAC OMA 2012.01 (5).

Preparation of Moist Test Portions
The high-level wheat flour concentration stock, prepared in gluten-free evaporated milk matrix, was diluted, using a stepwise dilution scheme, to prepare a gluten-contaminated matrix batch for each level being tested (0, 5, 10, and 20 mg/kg of gluten from wheat flour).The contaminated matrix batch went through the same mixing process (as the high concentration stocks) for 30 min.
A portion of 500 mL of the 20 mg/kg gluten concentration of evaporated milk matrix was prepared mixing the gluten-free evaporated milk matrix with the corresponding amount of highlevel wheat flour concentration extract stock.
The 10 mg/kg gluten concentration level of evaporated milk matrix was prepared mixing 250 mL of the uniform gluten free evaporated milk matrix with 250 mL of the high-level wheat flour concentration extract stock contaminated at 20 mg/kg of gluten.
The 5 mg/kg gluten concentration level of evaporated milk matrix was prepared mixing 250 mL of the uniform gluten-free evaporated milk matrix with 250 mL of the high-level wheat flour concentration extract stock contaminated at 10 mg/kg of gluten.
Six individual 0.5 mL test portions of the evaporated milk matrix batch at each concentration of gluten (0, 5, 10, and 20 mg/kg of gluten from wheat flour) were portioned out into 50 mL polypropylene flat-bottom test tubes.Once prepared, the test portions were refrigerated (4 C) until use.

Preparation of Gluten-Free Bread and Incurred Breads by the Independent Laboratory
A bread maker was used to make and cook the gluten-free bread and the incurred breads, and a dehydrator to remove the water of the final product: Bread maker: Bread Maker Homemade Deluxe Princess Food dehydrator: Food Dryer Proficook PC-DR 1116 To prepare the breads, the instructions of gluten-free bread mix below were followed: Ingredients: Gluten-free bread mix: 410 g Bakery yeast: 3.5 g Distilled water: 330 mL Sunflower oil: 50 mL Spiking material: adequate amount To calculate the amount of spiking material, the final weight of the product as the sum of gluten-free bread mix weight (410 g) and sunflower oil volume (50 mL$ 50 g), giving a total of 460 g, was considered. Procedure: (1) 205 g gluten-free bread mix was added to the bread pan.
(2) The spiking material was added to the bread pan, spreading it over the blank material.(3) Liquid ingredients (50 mL sunflower oil and 330 mL distilled water) were added to the bread pan.(4) 205 g gluten-free bread mix was added to the bread pan.
(5) 3.5 g bakery yeast was added to the bread pan.
To cook the bread mass, the specific program for gluten-free bread offered by the bread maker was followed.
Once the baking process was finished, the breads were cut into small pieces and frozen at À20 C overnight.
The following day, the material was ground while it was still frozen.Afterwards, the material was dehydrated for 48 h at 70 C using the food dehydrator.
After the dehydration process, the material was ground again and mixed using a food mixer.

Calibration Study
The calibration study was conducted in every extracted sample dilution (extracts prepared directly from wheat flour using the candidate method extraction mentioned in the GlutenTox ELISA Rapid G12 manual (Table 1) according to the expected amount of gluten.
Using the set of standards/calibrators supplied with the kit, a dose response curve was determined at every extracted sample dilution, using five (or six) concentrations over the concentration range of the kit with five replicates each.
The results are shown in Figures 1-8.The calibration study conducted with the GlutenTox ELISA Rapid G12 in all dilutions produced results where a trend of a nonrandom pattern was found in the higher analyte concentrations of each dilution.

Matrix Study With Wheat Flour (Non-Processed Foods)
The matrix study was performed to test the ability of the GlutenTox ELISA Rapid G12 test kit to determine the gluten-containing reference material (wheat flour) in each of the five selected food matrixes (soy flour, corn bread/biscuit, rolled oat, seasoning mix, and evaporated milk).Spikes were tested at four different levels of gluten (0, 5, 10, and 20 mg/kg).For each spike level, six blind-coded (individually extracted) test portions were analyzed by one technician using the GlutenTox ELISA Rapid G12 method.Test portions (replicates) were taken from a y = 0.8928x 2    Matrix Study With Barley and Rye Flours (Non-Processed Foods) The matrix study was performed to test the ability of the GlutenTox ELISA Rapid G12 test kit to determine the glutencontaining reference material (barley and rye flour) in each of the five selected food matrixes (soy flour, corn bread/biscuit, rolled oat, seasoning mix, and evaporated milk).Spikes were

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The evaluation of the same sample extracts for repeatability produced average values of 6.359% RSD at 10 mg/kg, and 4.014% RSD at 20 mg/kg spike level of gluten from barley and 16.829% RSD at 10 mg/kg, and 4.244% RSD at 20 mg/kg spike level of gluten from rye.For soy flour, a summary of results is presented in Table 3 and Figures 14 and 15     barley and 7.171% RSD at 10 mg/kg, and 14.206% RSD at 20 mg/kg spike level of gluten from rye.For corn bread, a summary of results is presented in Table 3  For evaporated milk, a summary of results is presented in Table 3 and Figures 22 and 23.
Incurred Matrix Testing With Wheat, Barley, and Rye Flours (Processed Foods) The incurred matrix study was conducted in the same fashion as the food matrix study but only on bread (gluten-free bread mix).Incurred bread matrixes were spiked, baked, and prepared by the independent laboratory.The uncooked matrixes were spiked at three concentrations of gluten (0, 20, and 30 mg/kg) with the target analyte (from wheat flour, barley flour, or rye flour) and then baked/cooked.The independent laboratory prepared the test portions for testing at the independent laboratory and Hygiena's laboratory to ensure both internal and independent validation studies were conducted using the same incurred test portions.
The GlutenTox ELISA Rapid G12 method was tested with 10 blank incurred test portions and 20 incurred test portions spiked at 20 mg/kg gluten (10 replicates) and 30 mg/kg gluten (10 replicates) with wheat flour.The same procedure was carried out when the target analyte was from barley flour or rye flour.
The results are shown in

LOD and LOQ Determinations of Spiked Matrixes (Unprocessed)
Data are submitted showing the LOD and LOQ of one sample of each of the gluten-free matrixes (blank test portions) tested 10 times.The LOD is expressed as the mean value of the negative sample determination (blank result) plus 3.3 SDs.LOD was determined to be 0.77 mg/kg (soy flour), 0.74 mg/kg (corn bread), 0.72 mg/kg (rolled oats), 0.77 mg/kg (seasoning mix), and 0.69 mg/kg (evaporated milk) by GlutenTox ELISA Rapid G12, with a mean LOD of 0.738 mg/kg across all matrixes tested.
The LOQ is expressed as the mean value of the negative sample determination (blank result) plus 10 SDs.
A summary of LOD and LOQ results is presented in Tables 5  and 6.
(a) Validation of the estimated LOQ (LOQ est ).-Each matrix was spiked with wheat flour at the LOQ est (1.59 mg/kg soy flour, 1.51 mg/kg corn bread, 1.57 mg/kg rolled oats, 1.70 mg/kg seasoning mix, and 1.46 mg/kg evaporated milk), and 10 test portions were tested to demonstrate acceptable precision, RSDr of less than 20%.

LOD and LOQ Determinations of Incurred Matrix (Processed Food)
Gluten-free bread matrix spiked with wheat, barley, and rye flours, separately, was baked and prepared by the independent laboratory.LOD and LOQ were also estimated in 30 blank test portions of the gluten-free incurred bread matrix provided by the independent laboratory to perform the incurred matrix study.The LOD is expressed as the mean value of the negative sample determination (blank result) plus 3.3 SDs.
LOD was determined to be 0.193 mg/kg (10 blank test portions for the incurred bread matrix study with wheat flour), 0.333 mg/ kg (10 blank test portions for the incurred bread matrix study with barley flour), and 0.215 mg/kg (10 blank test portions for the incurred bread matrix study with rye flour) by GlutenTox ELISA Rapid G12, with a mean of 0.247 mg/kg across all test portions tested.
The LOQ is expressed as the mean value of the negative sample determination (blank result) plus 10 SDs.
LOQ was determined to be 0.414 mg/kg (incurred bread matrix study with wheat flour), 0.845 mg/kg (incurred bread matrix study with barley flour), and 0.493 mg/kg (incurred bread matrix study with rye flour) by GlutenTox ELISA Rapid G12, with a mean LOQ of 0.584 mg/kg across all test portions tested.
A summary of LOD and LOQ results is presented in Table 7.

Selectivity Study
The selectivity study was performed to demonstrate that the GlutenTox ELISA Rapid G12 method does not produce positive results, cross-reactivity, when tested on common food ingredients that do not contain any gluten, and at the same time demonstrate this method's ability to detect target compounds (gluten from wheat, barley, and rye) without interference from the matrix when tested in the presence of gluten.The list of compounds recommended by Guidelines for Validation of Quantitative Gluten Methods, with Specific Examples for ELISA Assays was prescreened using the AOAC OMA 2012.01 method (5) to detect natural contamination prior to the study.Each compound was purchased from a local or online store and was tested as it normally would be consumed (raw or cooked), based on full-strength extracts.Guar gum and xanthan gum were diluted in rice flour due to the viscous nature of the compounds.
Individual test portions of each compound without contamination (blank test portions) were prepared.Using a high concentration stock spiking material in rice flour, individual test portions of each compound with 20 mg/kg gluten from wheat flour were also prepared.Blind-coded and randomized test portions (spiked and blank test portions) were tested once using the GlutenTox ELISA Rapid G12 test kit according to the method instructions.Any unspiked compounds that tested positive were required to be retested by testing six test portions of the compound with the GlutenTox ELISA Rapid G12 test kit.
Each blank compound and gluten spiked compound were tested once according to the GlutenTox ELISA Rapid G12 test kit package insert.The results are shown in Table 8.
All matrixes tested using the GlutenTox ELISA Rapid G12 test kit gave results <0.6 mg/kg gluten (<LOQ) for the cross-reactivity study except for the same matrixes that tested positive in the prescreening evaluation with the AOAC OMA 2012.01 (5): oat flour (2.79 mg/kg gluten), romano bean flour (6.06 mg/kg gluten), fava bean flour (9.82 mg/kg gluten), and lima bean flour (297 mg/kg gluten).These matrixes were not retested with the GlutenTox ELISA Rapid G12 test kit since they had already produced positive results in the prescreening evaluation with the AOAC OMA 2012.01 (5).However, further analysis was carried out using the same matrixes in bean format (and rolled oats) and grinding them in the laboratory before performing the tests to minimize the risk of gluten contamination.Now the ground matrixes tested produced results <0.6 mg/kg gluten (<LOQ) for the cross-reactivity study (Table 8).For the 20 mg/kg spike level, all matrixes tested using the GlutenTox ELISA Rapid G12 test kit gave positive results in the interference study, ranging from 14.82 mg/kg gluten (milk: whole, liquid) to 24.17 mg/kg gluten (yellow pea flour).

Intermediate Precision Study
Intermediate precision of GlutenTox ELISA Rapid G12 test method was examined using the design 2b (Figure 27) and the results are presented in Table 11.The validation statistics are shown in Tables 12, 13 and 14.
Incurred test material (gluten-free bread mix spiked with wheat flour) at 20 mg/kg spike level of gluten (middle level) was tested using three GlutenTox ELISA Rapid G12 test kit lots.Two operators conducted analysis on two days for each test kit lot.Each day, the assigned operator conducted analysis of two individually extracted test portions of test material, with the ELISA measurements performed in duplicate for each.
Single Lab Validation set from GlutenTox ELISA Rapid G12 test method was analyzed for validation statistics.The method validation design used 3 kit lots, 2 analysts over 2 days, with 2 test portions per sample and 2 ELISA per test portion.This is described as AOAC GFA Design design 2b, with modification that the end point determination (the ELISA measurement) was performed in duplicate (per GlutenTox instructions), that is, the design was run twice.Since 12 calibrations were performed, it was decided to analyze Analyst/Day/Calibration as a single confounded factor as a stand-in variable for these intermediate factors.This confounded "Calibration" factor was deemed to be nested within "Lot", so a fully nested analysis model was used here.This was a single test material at an approximate detection level of 12 mg/kg gluten.
In this study, for this matrix, the nested ANOVA table (Table 12) shows the greatest contribution to overall variance is the confounded factor -(Day/Analyst/Calibration) at 57.2% of the variance.The second highest was ELISA (error) at 29.5% of total variance.Interestingly, kit Lot was very small (0%) contributor to overall variance.The variance of the final method can be estimated by mathematically dividing the variances of the components by the number of replicates of the study.In this case, the overall RSD for the method is 10.33% (sd(i) ¼ 1.30) (Tables 12 and 13) which is mathematically reduced to 6.19% (by over 4%) (sd(i) ¼ 0,779) (Table 14) when the variance of the ELISA component (0.49945) is divided by the number of replicates (two ELISA wells per test portion) tested (obtaining a value of 0.249725 for the variance of the ELISA component).

Stability Study
Stability of GlutenTox ELISA Rapid G12 test kit was examined in rice flour matrix through accelerated studies based on the  Rice flour matrix spiked with wheat flour at three different spiked levels of gluten (0 mg/kg, 10 mg/kg and 20 mg/kg) were prepared using the same preparation techniques as used in the matrix study.For each spiked level of gluten and at each stability time point, 5 blind coded replicate test portions were analyzed using the GlutenTox ELISA Rapid G12 test method.
As accelerated stability provides only a rough estimate of shelf-life, real time data supporting the entire shelf life of the kit under normal storage conditions (2-8 C) was also performed in the same matrix (rice flour) and spiked levels of gluten (0 mg/kg, 10 mg/kg and 20 mg/kg) and following the same procedure as the accelerated study but at different specified time points (3 months, 9 months, and 15 months).
Accelerated and real time stability results are presented in Table 15.
Performance Tested Methods SM program: PTM Validation of the GlutenTox ELISA Rapid G12 Kit in Select Foods, version 10, September 27, 2021, used in the production of gluten-free products.Four compounds that had tested positive in the prescreening evaluation with the AOAC OMA 2012.01 (5) (oat flour, romano bean flour, fava bean flour, and lima bean flour) also tested >LOQ with the GlutenTox ELISA Rapid G12 test kit and were not retested.To determine if the positivity of these matrixes was due to gluten contamination during the manufacturing process or to a crossreaction, further analysis was carried out using the same matrixes in bean format (and rolled oats) and grinding them in the laboratory before performing the tests to minimize the risk of a gluten contamination.Definitively, the GlutenTox ELISA Rapid test kit did not show cross-reactivity with these matrixes.Therefore, it can be confirmed that the previous positive results were due to gluten contamination.The GlutenTox ELISA Rapid G12 assay also did not show any interference, when tested with the required compounds for testing in the presence of gluten.No unexpected results were obtained (the lima bean matrix included in the interference study was that in bean format and subsequently ground).
The GlutenTox ELISA Rapid G12 test kit performed as expected when six additional wheat flour varieties were tested in rice flour, and positive results were obtained in all wheat cultivars analyzed.However, with the einkorn wheat flour (Triticum monococcum) variety, a recovery result lower than expected was obtained.Further studies are needed to determine if this is due to a lower gluten: protein ratio.
The lot-to-lot data (from the intermediate precision study), the accelerated stability data (8 days, 32 days, and 40 days at 25 C), and the real-time stability data (3 months, 9 months, and 15 months at 2-8 C) showed evidence that the GlutenTox ELISA Rapid G12 method is stable and can be consistently manufactured with reproducible quality.
Robustness data indicated that the GlutenTox ELISA Rapid G12 assay remained unaffected by minor variations in procedural parameters.
The GlutenTox ELISA Rapid G12 test kit performed as expected in the selected food matrixes (gluten-free soy flour, corn bread, seasoning mix, rolled oats, and evaporated milk), spike levels of gluten with wheat flour, and in both Hygiena (method developer) and the independent laboratory (only the corn bread and seasoning mix matrixes were tested), obtaining comparable results.
In all matrixes tested at different spike levels with barley and rye flours, the GlutenTox ELISA Rapid G12 assay performed as expected (meeting performance claims for recovery and repeatability, mainly with barley flour) or showing slight (<28%) to moderate (46% or 85%) overestimation depending on the matrix, source of gluten contaminant, and gluten concentration.
Results obtained in the method developer incurred matrix study with wheat, barley, and rye flours indicate that the assay performed as expected (meeting performance claims for recovery and repeatability, mainly with wheat and rye flours) or showing slight (25% or 37%) to moderate (49%) overestimation depending on the source of gluten contaminant and gluten concentration.These data are comparable to those obtained in the incurred sample study of the independent laboratory where the GlutenTox ELISA Rapid G12 method performed as expected with wheat and rye flours and showed a slight (11%) to moderate (77%) overestimation with barley at 20 mg/ kg and 30 mg/kg spike levels of gluten, respectively.
Nevertheless, this occasional overestimation of gluten from barley or rye is a less important factor in gluten analysis for the people with celiac disease, since possible problems from false negatives or underestimations could be much worse.No false negative results were observed in the entire validation study.
The GlutenTox ELISA Rapid G12 assay performed as expected in the calibration study in all dilutions.To minimize the trend of a nonrandom pattern found in the higher analyte concentrations of each dilution, a suitable dilution should be made according to the expected amount of gluten in the sample.
The intermediate precision study demonstrated that the design 2b and the contribution of the analyst/day/calibration as a single confounded factor to the variance were appropriate when the GlutenTox ELISA Rapid G12 assay was tested with the incurred bread matrix.
In this study, the overall RSD for the method was in accordance with the acceptance criteria, and even was mathematically reduced by over 4% when the variance of the ELISA component was divided by the number of replicates tested (two ELISA wells per test portion).
The overall validation of the estimated LOQ (LOQ est ) of the GlutenTox ELISA Rapid G12 test kit by the method developer in the selected matrixes performed as expected, showing an excellent correlation with the overall LOD-LOQ est (according to the standard deviation of blank samples).These results are in line and are consistent with the LOD and LOQ values obtained from the independent laboratory (calculated from the linear regression model) using three matrixes and four spike concentration levels of gluten from wheat flour (LOD ¼ 0.4 mg/kg gluten and LOQ ¼ 1.2 mg/kg gluten).

Conclusions
The GlutenTox ELISA Rapid G12 test kit is a quick and easy-to-use method for the detection and quantification of gluten concentration in food and incurred matrixes from wheat, barley, and rye flours.
The method is specific and reliable and provides sensitive and accurate test results, showing occasional slight to moderate overestimation depending on the matrix and gluten concentration from barley and rye flours, and it should be granted PTM certification.
The GlutenTox ELISA Rapid G12 test kit is a stable and costeffective kit recommended for laboratories and industry.The instructions for use include information about the preparation of different dilutions depending on the expected gluten content of the sample.
(a) Analyte(s).-Gluten from wheat, barley, and rye flour.(b) Matrixes.-Soyflour, corn bread, seasoning mix, rolled oats, evaporated milk, and gluten-free baked bread.(c) Summary of validated performance claims.-TheGlutenTox ELISA Rapid G12 test kit is designed to detect and quantify gluten in processed and non-processed foods listed above at a range of 0.6-200 mg/kg gluten.This range of quantitation is suitable for proposed gluten-free monitoring in the United States and is compliant with current EU regulations and Codex Alimentarius definitions.Definitions (a) Repeatability.-Precision under repeatability conditions.(ISO 5725-2) (Repeatability conditions: Conditions where independent test results are obtained with the same method on equivalent test items in the same laboratory by the same operator using the same equipment within short intervals of time (3).(b) Reproducibility.-Precisionunder reproducibility conditions.(ISO 5725-2) (Reproducibility conditions: Conditions where independent test results are obtained with the same method on equivalent test items in different laboratories with different operators using separate instruments (3).(c) Intermediate precision.-Precisionunder intermediate conditions.(ISO 3534-2).(Intermediate precision conditions: (i) Standard deviation of repeatability.-sr ¼ Relative standard deviation of repeatability.-RSDr ¼ [s r /mean cand ] Â 100 (k) LOD.-Mean þ 3.3s r , or X0þ3:3ðs b Þ 1À1:65m

( a )
Take a representative sample of the food and mill and/or triturate it thoroughly.(b) Homogenize by shaking the sample by hand for 1 min.(c) Weigh 0.5 g of the sample into a test tube.(d) Add 5 mL of extraction solution.Close the tube and mix vigorously using a vortex mixer for 30 s. (e) Depending on the complexity of the sample matrix and whether the food sample has been processed by heat or not, follow one of the two options below.(1) Non-heat-processed samples with simple matrix composition (a) Incubate the sample at room temperature (15-25 C/59-77 F) for 40 min with mild agitation (for example, using a tube rotator).(2) Heat-processed sample and/or complex matrix composition (incurred bread) (a) Incubate the sample at 50 C (122 F) in a water bath for 40 min; periodically mix the sample by inverting or vortexing the tube.(f) Centrifuge the suspension at 2500 Â g for 10 min and transfer the supernatant to a clean tube.Extraction of liquid samples: Liquid samples without emulsions or solids do not require intensive extraction.Manual shaking or vortexing is enough, and the final step of centrifugation is not required.(a) Shake the sample to homogenize.(b) Add 0.5 mL of sample in a test tube.(c) Add 4.5 mL of extraction solution.Close the tube and shake for 2 min manually or using a vortex mixer.Analysis (a) Allow all reagents and test strips, except GlutenTox A1-HRP conjugated antibody, to come to room temperature (15-25 C/59-77 F) before testing.(b) All assay reactions should be performed at least in duplicate.(c) Prepare the appropriate dilutions of the extracted, clarified samples using the included Dilution Solution in polypropylene vials.A final volume of 300 mL is enough for the analysis of each sample.Extracted sample dilutions should be analyzed as soon as possible and any unused material discarded.(d) Depending on the expected gluten content of the sample,

( a )
Determine average absorbance values for the replicates of each condition.(b) Prepare a standard curve by plotting gliadin concentrations of each GlutenTox standard (y-axis) versus the respective absorbance values (x-axis) obtained from the calibration standards using appropriate software (for example, Excel).Please contact Hygiena Diagno ´stica España to obtain an Excel template.(c) Calculate the equation that defines the standard curve by second-order polynomial regression using suitable software (for instance, Excel).(d) Enter the sample absorbance values obtained for each sample into this equation to obtain gliadin concentrations of the sample dilutions.

Table 1 .
GlutenTox Evaporated milk.-Theevaluation of samples extracts for Figure 6.GlutenTox ELISA Rapid G12-Calibration study: Residual plot 1:100 dilution.(b)Cornbread.-Theevaluation of samplesextracts for recovery produced average values of <LOQ recovery at 0 mg/kg spike level of gluten, 123.95% recovery (6.20 mg/kg mean value) at 5 mg/kg spike level, 97.70% recovery (9.77 mg/kg mean value) at 10 mg/kg spike level, and 100.91% recovery (20.18 mg/kg mean value) at 20 mg/kg spike level.The evaluation of the same sample extracts for repeatability produced average values of 11.79% RSD at 5.0 mg/kg, (d) Rolled oats.-Theevaluation of samples extracts for recovery produced average values of <LOQ recovery at 0 mg/kg spike level of gluten, 113.96% recovery (5.70 mg/kg mean value) at 5 mg/kg spike level, 91.31% recovery (9.13 mg/kg mean value) at 10 mg/kg spike level, and 104.26% recovery (20.85 mg/kg mean value) at 20 mg/kg spike level.The evaluation of the same sample extracts for repeatability produced average values of 6.31% RSD at 5.0 mg/kg, recovery produced average values of <LOQ recovery at 0 mg/kg spike level of gluten, 63.00% recovery (3.13 mg/kg mean value) at 5 mg/kg spike level, 66.00% recovery (6.58 mg/kg mean value) at 10 mg/kg spike level, and 72.00% recovery (14.31 mg/kg mean value) at 20 mg/kg spike level.The evaluation of the same sample extracts for repeatability produced average values of 1.00% RSD at

Table 2 .
GlutenTox ELISA Rapid G12-Food matrix study with wheat flour from candidate and from independent laboratory (analyst 1 and analyst 2) Soy flour.-Theevaluation of samples extracts for recovery produced average values of <LOQ recovery at 0 mg/kg replicate) test portions were analyzed by one technician using the GlutenTox ELISA Rapid G12 method.Matrixes were prepared and contaminated in the same manner as the wheat flour spiked test portions, using the appropriate conversion factors for barley (0.75) and rye (0.48).The matrix study determines the bias, recovery, and repeatability precision of the GlutenTox ELISA Rapid G12 test kit.(a) spike level of gluten, 153.736% recovery (15.374 mg/kg mean value) at 10 mg/kg spike level gluten from barley and 182.064% recovery (18.206 mg/kg mean value) at 10 mg/kg spike level gluten from rye, 158.897% recovery (31.779 mg/ kg mean value) at 20 mg/kg spike level gluten from barley and 176.878% recovery (35.376 mg/kg mean value) at 20 mg/kg spike level gluten from rye.

Table 3 .
GlutenTox ELISA Rapid G12-Food matrix study with barley and rye flours from candidate level gluten from barley and 277.861% recovery (55.572 mg/kg mean value) at 20 mg/kg spike level gluten from rye.The evaluation of the same sample extracts for repeatability produced average values of 15.675% RSD at 10 mg/kg, and 13.738% RSD at 20 mg/kg spike level of gluten from mg/kg spike level gluten from rye, 219.441% recovery (43.888 mg/kg mean value) at 20 mg/kg spike level gluten from barley and 181.157% recovery (36.231 mg/kg mean value) at 20 mg/kg spike level gluten from rye.The evaluation of the same sample extracts for repeatability produced average values of 7.516% RSD at 10 mg/kg, and 9.071% RSD at 20 mg/kg spike level of gluten from barley and 1.008% RSD at 10 mg/kg, and 2.628% RSD at 20 mg/ kg spike level of gluten from rye.
(e) Evaporated milk.-Theevaluation of samples extracts for recovery produced average values of <LOQ recovery at 0 mg/kg spike level of gluten, 187.370% recovery (18.737 mg/kg mean value) at 10 mg/kg spike level gluten from barley and 181.292% recovery (18.129 mg/kg mean value) at 10

Table 4
The evaluation of the same incurred sample extracts for repeatability produced average values of 8.05% RSD at 20 mg/kg spike level and 8.97% RSD at 30 mg/kg spike level of gluten.

Wheat incurred bread 0 mg/kg Barley incurred bread 20 mg/kg Barley incurred bread 30 mg/kg mg
/kg of gluten Figure 25.GlutenTox ELISA Rapid G12-Incurred matrix study with barley flour: Regression analysis per concentration.

Table 4 .
GlutenTox ELISA Rapid G12-Incurred matrix study from candidate and from independent laboratory (analyst 1 and analyst 2)

Table 10 .
GlutenTox ELISA Rapid G12-Robustness study Figure 27.Design 2b.Lot: test kit lot, TP: test portion, E: ELISA measurement.Design 2b can be used to estimate intermediate precision, repeatability, ELISA variance, and lot-to-lot product consistency.

Table 11 .
GlutenTox ELISA Rapid G12-Intermediate precision study (design 2b) in the incurred matrix from candidate Recovery mg/kg Recovery mg/kg Recovery mg/kg Recovery mg/kg Recovery mg/kg Recovery mg/kg Recovery mg/kg Recovery mg/kg Recovery mg/kg Recovery mg/kg Recovery mg/kg Recovery