hygiena KIT3005 GlutenTox SticksPlus Owner’s Manual

KIT3005 GlutenTox SticksPlus

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Specifications

• Product: Gluten detection kit for foods, drinks, and working
  surfaces

• Product No.: KIT3005

• Manufacturer: www.hygiena.com

Product Information

This gluten detection kit is designed for the semi-quantitative
determination of gluten in various food products, beverages, and
working surfaces. It uses the anti-gliadin G12 antibody to
specifically recognize the 33-mer peptide, the most immunogenic
fraction of gluten. The kit includes Universal Gluten Extraction
Solution (UGES) for efficient extraction of gluten from different
types of food matrices.

Test Basis

The GlutenTox Sticks Plus kit can be used to test a wide range
of food compositions and processing levels, from raw materials to
heat-processed foods. It can also be employed for surface testing
to ensure that surfaces are suitable for producing gluten-free
products. The kit aids in routine monitoring of gluten presence,
compliance with Hazard Analysis and Critical Control Points (HACCP)
programs, and proper labeling.

Supplied Materials

• GlutenTox Sticks (25 sticks in a tube)
• Universal Gluten Extraction Solution (UGES) – 250 mL
• Dilution Solution – 30 mL
• Microtiter plate strips (4 strips x 8 wells)
• Instructions for use

Product Usage Instructions

1. Sample Preparation

Prepare the sample according to the specific guidelines for the
type of food or surface being tested. Ensure the sample is
representative and properly collected.

2. Extraction Process

Use the Universal Gluten Extraction Solution (UGES) provided in
the kit to extract gluten from the sample matrix. Follow the
extraction procedure detailed in the instructions for use.

3. Testing Procedure

Follow the step-by-step instructions provided in the manual to
conduct the gluten detection test using the GlutenTox Sticks Plus.
Ensure proper handling and timing during the test process.

4. Interpretation of Results

Observe the appearance of a BLUE line on the test strip
post-testing. The presence of this line indicates a valid test with
proper sample volume, flow, and conjugate particle release. If the
BLUE line does not appear, consider the test invalid.

FAQ

Q: Can this kit be used for hydrolyzed sources of gluten?

A: No, this kit is not intended for hydrolyzed sources of
gluten.

Q: What should be done if the BLUE line does not appear after

testing?

A: If the BLUE line does not appear, consider the test invalid
and repeat the testing process with a new sample following proper
procedures.

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Gluten detection kit for foods, drinks and working surfaces
Product No. KIT3005

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Table of Contents
1. Intended Purpose ……………………………………………………………………………………………………………………………. 3 2. Introduction……………………………………………………………………………………………………………………………………. 3 3. Test Basis ……………………………………………………………………………………………………………………………………….. 3 4. Supplied Materials…………………………………………………………………………………………………………………………… 5 5. Necessary Materials Not Supplied …………………………………………………………………………………………………….. 5 6. Storage Conditions and Stability ……………………………………………………………………………………………………….. 5 7. Precautions…………………………………………………………………………………………………………………………………….. 6 8. Sample Preparation (Food, Beverages and Other Consumer Products) ………………………………………………….. 6
8.1 Solid Samples …………………………………………………………………………………………………………………………. 6 8.2 Liquid Samples ……………………………………………………………………………………………………………………….. 7 9. Test Implementation for Extracted Samples……………………………………………………………………………………….. 8 10. Interpretation of Results for Food Samples ………………………………………………………………………………………… 9 11. Surface Analysis…………………………………………………………………………………………………………………………….. 10 12. Interpretation of Results for Surface Analysis……………………………………………………………………………………. 11 12.1 Final Reading of the Stick at 10 Minutes…………………………………………………………………………………… 11 12.2 Initial Reading of the Stick at 4 Minutes …………………………………………………………………………………… 11 13. Quality Control ……………………………………………………………………………………………………………………………… 11 14. Analytical Features ………………………………………………………………………………………………………………………… 11 14.1 Sensitivity …………………………………………………………………………………………………………………………….. 12 14.2 Sensitivity in Surface Analysis …………………………………………………………………………………………………. 12 14.3 Specificity …………………………………………………………………………………………………………………………….. 12 14.4 Internal Validation ………………………………………………………………………………………………………………… 12 15. Intellectual Property………………………………………………………………………………………………………………………. 14 16. References……………………………………………………………………………………………………………………………………. 14

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1. Intended Purpose
GlutenTox® Sticks Plus is a rapid immunochromatographic test for the detection of gluten*, which is harmful for celiac disease sufferers, in food and on surfaces. This kit is recommended for consumers, commercial kitchens and industry.

• Not for hydrolyzed sources of gluten.
  2. Introduction
  Celiac disease is 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 is caused by an inappropriate response of the immune system to gluten (a mix of proteins found in cereals) from wheat, barley, rye and, to a lesser extent, from oat [1 and 2], leading to diarrhea, vitamin and mineral deficiencies, anemia and thin bones (osteoporosis). Celiac disease affects people of all ages.
  Currently, the only treatment for celiac disease sufferers is a strict, lifelong gluten-free diet that presents great difficulties because gluten, in addition to being present in many foods, may also be found in food additives and preservatives.
  According to the Codex Alimentarius Commission and the EC Regulation 41/2009 on the composition and labeling of foodstuffs suitable for people intolerant to gluten, food can be considered “gluten-free” if its gluten content does not exceed 20 parts per million (ppm).
  Milligrams of gluten per kilogram of food.
  3. Test Basis
  GlutenTox Sticks Plus is an immunochromatographic (lateral flow) test for the semi-quantitative determination of gluten in foods with different compositions and levels of processing, from raw materials to heat-processed food, beverages and other consumer products. It is based on the anti-gliadin G12 antibody, which specifically recognizes the 33-mer peptide, the most immunogenic fraction of gluten [3].
  GlutenTox Sticks Plus can also be used for surface testing to confirm that these surfaces are suited for use in the production of gluten-free products. This rapid test is useful in routine monitoring of gluten presence to guarantee that products comply with a program of Hazard Analysis and Critical Control Points (HACCP) and to ensure proper labeling. It also allows quick decisions and corrective actions in case there is any risk of contamination along the production chain.
  In all methods used for gluten analysis in a given sample, the gluten first has to be extracted from the sample’s matrix. Extraction is one of the most critical points of the testing process. The extraction solution provided in this kit, Universal Gluten Extraction Solution (UGES), is suited for all types of food thanks to the combination of denaturing agents, reducing agents and solubilizers.
  For the analysis of food containing polyphenols, including tannins, such as chocolate, tea, coffee, wine, purple corn and corn fiber, soy, berries, etc., it is necessary to use a special additive (not included in this kit, see section 5) that prevents the interference of the above-mentioned compounds in the extraction process (see Figure 1). The same applies in the case of cosmetic products with antioxidant compounds such as vitamins A, C and E, carotenes, carotenoids, etc.

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After the extraction, during the detection step, the gluten in the sample reacts first with the anti-gliadin G12 antibody [3] conjugated to red-colored particles, previously placed in the stick. The resulting complexes spread by capillarity through the stick and react with a second anti-gliadin antibody, also previously immobilized on the stick. If the result is positive, a RED line appears in the result zone of the stick. The absence of the RED line indicates a negative result. Whether or not gluten is present, the sample moves through the stick up to the control region where, if the test was properly performed, a BLUE line will appear, due to the accumulation of blue- colored particles also included in the stick.
The presence of this BLUE line indicates that 1) the sample volume was enough, 2) the sample flow was appropriate and 3) the conjugate particles included in the test were properly released. If the BLUE line does not appear, the test should be considered invalid.

GlutenTox Sticks Plus

Figure 1. Scheme of Use for GlutenTox Sticks Plus. 4

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4. Supplied Materials
GlutenTox Sticks (25 sticks in a tube) Universal Gluten Extraction Solution (UGES)
(250 mL)

Dilution Solution (30 mL) Microtiter plate strips (4 strips x 8 wells) Instructions for use

5. Necessary Materials Not Supplied
Positive Control (wholegrain oat flour contaminated with gluten, 10 g) (Product no. ASY3052)
Negative Control (corn flour, 10 g) (Product no. ASY3051)
Analytical scale (accurate to 0.1 g)
Thermostatic bath (not necessary for non-heatprocessed samples with simple matrix composition)

Capped centrifuge test tubes (>10 mL) Test vials (1.5 ­ 2 mL) Pipettes and disposable tips Disposable gloves Tube rotator Watch/chronometer Centrifuge (optional) Vortex mixer (optional)

For testing food containing polyphenols (including tannins) and cosmetics containing antioxidants, please acquire the Polyphenol Pack (KIT3008)*, available from Hygiena®. This pack contains: Special polyphenol additive (25 g). Positive Control containing polyphenols (cocoa powder with gluten, 10 g). Negative Control containing polyphenols (gluten-free cocoa powder, 10 g). Important: Foods rich in polyphenols or tannins are: chocolate, tea, coffee, wine, purple corn and corn fiber, soy, berries, legumes like chickpeas or lentils, etc. Important: The most common antioxidants in cosmetic products are vitamins A, C and E, carotenes, carotenoids, etc.

• For more information contact your supplier.

6. Storage Conditions and Stability
For optimal test performance, GlutenTox Sticks Plus must be stored in its original packaging, at 15 to 25 °C (59 to 77 °F) and used before the expiration date printed on the label.
WARNING: The tube with the sticks should not be opened until the time of use. Once the seal is broken, keep the tube with the sticks tightly closed at room temperature (15 to 25 °C/59 to 77 °F). To avoid water condensation, do not refrigerate the tube after opening. Never freeze it.

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7. Precautions
Only for testing food, beverages, other consumer products and surfaces. Do not ingest any of the solutions (liquids) and/or additives of the kit. Do not use after the expiration date. The use of non-powdered disposable gloves is recommended. Manipulate the sticks with gloves or washed hands and do not touch the white end. If a sample is heterogeneous (e.g., a salad), make sure to take a representative part of each ingredient, and
mix them to make a homogenous sample. If the gluten in the sample is unevenly distributed and you do not do this, a false negative could be obtained.
8. Sample Preparation (Food, Beverages and Other Consumer Products)
8.1 Solid Samples
1. Homogenize, mill and/or triturate the sample. 2. Weigh 1 g of sample and add it to a test tube.
Important: If the sample, solid or liquid, contains polyphenols, tannins (e.g., chocolate) or antioxidants, weigh and add 1 g of special polyphenol additive (KIT3008) to the sample tube and mix it vigorously to achieve complete homogenization of the mixture. 3. Add 10 mL of Universal Gluten Extraction Solution (UGES) to each sample, including positive and negative control samples. Close the tube and mix to homogenize (for example, using a vortex mixer). 4. Depending on the complexity of the sample matrix and whether the food sample has been processed by heat or not, follow one of the options below (see Figure 2): a) Non-heat processed samples with simple matrix composition:
Incubate the sample at room temperature (15 to 25 °C, 59 to 77 °F) for 40 minutes with a tube rotator. b) Heat-processed samples and/or with complex matrix composition or samples containing polyphenols,
tannins or antioxidants: Incubate the sample at 50 °C (122 °F) in a water bath for 40 minutes, shaking the tube periodically by tipping it over or using a vortex mixer. Important: If the type of sample is difficult to determine, we recommend heating at 50 °C (122 °F) (option b) to facilitate the extraction. 5. Allow separation of solids by settling or centrifugation (10 min at 2,500 x g). 6. Transfer the clarified supernatant to a clean tube. Important: Once extracted, the samples must be analyzed as quickly as possible.

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Figure 2. Scheme of the Extraction Procedure of Solid Samples.
8.2 Liquid Samples
Important: Liquid samples with polyphenols, tannins or antioxidants must be extracted according to point 8.1. Solid samples. Liquid samples such as milk, juices, soft drinks, organic drinks (soy, rice, oat, spelt drinks), beers and broths do not require intensive extraction. Manually shaking for 1 ­ 2 minutes is sufficient, and the extracts do not require a centrifugation or settling step. 1. Add 1 mL of sample to a test tube. 2. Add 9 mL of Universal Gluten Extraction Solution (UGES). Close the tube and mix to homogenize (for
example, using a vortex mixer). 3. Shake the sample for 1 ­ 2 minutes, manually or using a vortex mixer.
Important: Once extracted, the samples must be analyzed as soon as possible.

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9. Test Implementation for Extracted Samples
1. Bring the extracted samples, controls, Dilution Solution and the tube with the GlutenTox Sticks to room temperature (15 to 25 °C, 59 to 77 °F).
2. Dilute the sample with the Dilution Solution in test tubes or vials. A final volume of 900 ­ 1000 L is sufficient to perform the test.

Table 1. Dilutions Table.

Dilution
1:25 1:75 1:150 1:250 1:750

Rounded Gluten Detection Limit (ppm)
3 10 20 30 100

Real Gluten Detection Volume of Extracted

Limit (ppm)

Sample (µL)

3.5

40

10.5

13.3

21

6.6

35

4

105

1.3

Volume of Dilution Solution (µL) 960 986.7 993.4 996 998.7

If the expected amount of gluten in the sample is unknown, we recommend testing with the lowest dilution (i.e., dilution 1:25), allowing maximum sensitivity. In case of a positive result (appearance of RED line), the test can be repeated with a greater dilution for a semi-quantitative estimation of the gluten concentration in the sample (see Section 14, Analytical Features).
Important: In samples with polyphenols, tannins or antioxidants, in which the special polyphenol additive is added in the extraction step, the minimum dilution allowed is 1:75. Never perform at lower dilutions (i.e., dilution 1:25) since the presence of the special polyphenol additive affects the proper performance of the stick if the dilution is less than 1:75.
Important: In samples with high levels of fat, avoid taking the upper layer that contains the fat.
Important: The diluted samples must be analyzed as quickly as possible, and the remaining material should be discarded.
3. Place 100 L of the diluted sample in a well of the microtiter strip supplied in the kit.
4. Open the tube with GlutenTox Sticks, take out the number of sticks necessary and close the tube immediately.
5. Introduce the white end of the stick vertically into the well with the diluted sample.
6. Wait 10 minutes and read the result on the stick.

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Figure 3. Procedure to Follow for Sample Analysis.
10. Interpretation of Results for Food Samples
NEGATIVE: A single BLUE line (control line) appears in the central part of the stick (control zone).
POSITIVE: In addition to the control line (BLUE), a RED line (result line) appears in the result zone. See Table 2 to read the result depending on the selected Detection Limit. Important: The intensity of the red line in the result zone will vary depending on the gluten concentration in the sample.
INVALID: The control line (BLUE) does not appear, whether or not the result line (RED) appears. The most common causes of an invalid result are using an insufficient (<100 L) volume of sample in the microtiter well, performing an incorrect procedure or deterioration of the kit reagents. In the case of an invalid result, it is necessary to revise the procedure and repeat the experiment with a new test. If the problem persists, please contact your supplier.

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11. Surface Analysis
1. Bring the Dilution Solution and the tube with the GlutenTox Sticks to room temperature (15 to 25 °C, 59 to 77 °F). 2. Place 100 L of the Dilution Solution in a well of the microtiter strip supplied in the kit. 3. Rub the cotton wool side of the white end of the stick against five areas of 1 cm2 or along a line of 1 x 5 cm
(see Figure 4). 4. After the stick has been rubbed on the surface to be analyzed, introduce its white end into the well. 5. Wait 10 minutes and read the result on the stick.
Figure 4. Procedure for Surface Analysis.

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12. Interpretation of Results for Surface Analysis
12.1 Final Reading of the Stick at 10 Minutes
12.2 Initial Reading of the Stick at 4 Minutes Optionally, an initial reading of the stick after 4 minutes can be done to obtain a semiquantitative measurement. If a red line appears already after 4 minutes, the surface area tested had a gluten content equivalent to >10 ppm.

13. Quality Control
An internal procedural quality control is included in the test. The blue line in the control zone is a built-in feature that indicates both a sufficient volume and a correct flow of the sample, with proper release of the conjugate particles. In addition, this manual includes instructions for use of Positive and Negative Controls (see point 8.1.2 and onwards) to confirm a correct test performance. If the test procedure was conducted properly, these control materials must provide clear positive and negative results, respectively, at any tested dilutions.
14. Analytical Features
Different assays have been carried out to characterize the main analytical parameters of the test, i.e., sensitivity and specificity.

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14.1 Sensitivity
The detection limit of GlutenTox Sticks Plus is 3 ppm of gluten. This value was obtained using different solutions of known concentrations of gliadin (one of the proteins in gluten) in the Dilution Solution.
For food, beverages and other consumer products, the detection limit of the test in ppm of gluten will depend on the dilution factor applied to the sample, once extracted.
Table 2 outlines the dilution to be carried out according to the level of gluten to detect.

Table 2. Detection Limit in ppm of Gluten According to the Dilution of the Test.

Test Result

Positive Negative

1:25 >3 ppm <3 ppm

Detection Limit/ppm Gluten

Dilutions

1:75

1:150

1:250

10 ppm

20 ppm

30 ppm

<10 ppm

<20 ppm

<30 ppm

1:750 >100 ppm <100 ppm

14.2 Sensitivity in Surface Analysis
When testing surfaces, the result obtained with the test indicates the presence or absence of gluten on the analyzed surface; it cannot be directly extrapolated into any value of gluten in ppm.
By analyzing five areas of 1 cm2 or a rectangle of 1 x 5 cm a minimum of 40 ng/cm2 is detected. If the test result is negative, it can be “estimated” that for an analyzed working surface of 1000 cm2 (40 cm x 25 cm), working with a food mass of 1 kg, the final product will have less than 0.04 ppm (0.04 mg gluten/kg of food). This quantity is about 500 times less than the quantity recommended by the European norms established at 20 ppm (20 mg gluten/kg of food).
This means that the method has a large safety margin and that its use provides a guarantee to clients, celiac associations and food safety inspectors [4].
14.3 Specificity
This test can specifically detect the presence of the toxic fraction of the prolamins of wheat (gliadin), rye (secalin), barley (hordein) and some varieties of immunogenic oats (avenin) that can therefore be harmful for celiac patients [2]. However, when the samples contain celiac-safe foods like rice, corn, soy, buckwheat, sesame, millet, teff, quinoa and amaranth, no positive signal is observed.
14.4 Internal Validation
To ensure the test’s performance with all types of food and other materials such as cosmetics and personal care products, as well as with common working surface materials, a broad range of commercial products has been tested. After analyzing the samples with GlutenTox Sticks in all types of matrices tested (see Tables 3 and 4) the results were satisfactory and consistent with the gluten contents determined with the approved method of Codex Alimentarius. Results also showed the applicability of the test to common working surface materials (Table 5).

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Table 3. Food Samples Validated with GlutenTox Sticks Plus.

Group Flour and semolina
Milk products
Baked and cereal products
Meat products
Fishery products Vegetables
Broth, soups, creams and dry mixes
Sauces, dressing, spices and condiments Sugars
Prepared meals and dishes Fatty foods Acidic foods Beverages

Tested Samples
Corn flour, precooked corn flour, corn semolina, rice flour, wheat flour, buckwheat flour
Cow milk, milk with soluble fiber, milk with cereals, flavored or natural yogurt, cheese spread, shredded cheese blend
Toast, bread sticks, biscuits (Rich tea), chocolate cookies, Madeleine, cake, cornflakes, pastas, corn pancakes, rice cakes, spelt cake, snacks
Minced turkey, minced chicken, turkey sausage, chicken nuggets, pork sausages, chorizo, pork liver pâté
Cod and hake
Lettuce mix, fried vegetables
Vegetable broth, chicken rice soup, dehydrated vegetable soup, stock cubes, vegetable soup, peanut butter
Yogurt salad dressing, ketchup, soy sauce, salad dressing, garlic powder, paprika powder, cooking cream
Glucose syrup, powdered sugar
Meatballs in sauce with peas, meat ravioli in egg dough, bean stew
Olive oil, sunflower oil, butter, margarine, cream
Tomate sauce, wine vinegar, apple cider vinegar, lemon juice
Water, milk, fruit juices, beer, soy drinks, rice drinks, oat drinks, soft drinks

Table 4. Non-food Samples Validated with GlutenTox Sticks Plus.

Group Personal care products
Cosmetics Others

Tested Samples Bath gel, shampoo, deodorant, toothpaste, mouthwash
Creams (face, body and hands), cleanser, lip balm Pet food (dry food, wet food), cleaning products, drugs (tablets,
capsules and syrups)

Group Surfaces

Table 5. Surfaces Validated with GlutenTox Sticks Plus. Tested Samples
Stainless steel, rubber, painted wood

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15. Intellectual Property
The immunoreagents used in this kit are commercialized under the exclusive license for biological material from the Spanish National Research Council (CSIC).
16. References
1. Shan L., et al. (2002) Structural basis for gluten intolerance in celiac sprue. Science, 297:2275 ­ 2279. 2. Comino I. et al (2011) Diversity in oat potential immunogenicity: basis for the selection of oat varieties with
no toxicity in coeliac disease. Gut, 60:915 ­ 922. 3. Morón B., 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. 4. Síglez M.A., et al. (2010) Método de detección de gluten en superficies. Alimentaria, 411:67 ­ 70.

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GlutenTox Sticks Plus

Hygiena Camarillo, CA 93012
USA diagnostics.support@hygiena.com
Manufactured by Hygiena Diagnóstica España S.L.
P. I. Parque Plata Calle Cañada Real 31 ­ 35 41900, Camas (Sevilla), Spain
www.hygiena.com
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