AlerTox Sticks Walnut: Product Instructions
For the qualitative detection of walnut antigens in food, kitchens, and production facilities.
1. Intended Use
The AlerTox® Stick Walnut test kit is a rapid immunoassay lateral flow test kit for the qualitative detection of walnut antigens in food, kitchens, and production facilities. Samples prepared according to these instructions can be tested with AlerTox Sticks for Almond, Crustacean, Fish, Hazelnut, Mustard Seed, Sesame, and Walnut kits, but not with other AlerTox Sticks test kits. Read all instructions before beginning testing.
2. Introduction
English walnut or common walnut (Juglans regia) is a tree nut belonging to the Juglandaceae family. Walnut allergy is the most common tree nut allergy, with an estimated incidence of 0.1 - 1%. Walnut allergy can manifest in a variety of symptoms, ranging from mild oral allergy syndrome or hives to severe, life-threatening systemic reactions such as anaphylactic shock or asthma.
In the United States, the Food Allergen Labeling and Consumer Protection Act (FALCPA) identifies tree nut allergy as one of the major food allergies, requiring packaging to declare the presence of specific types of nuts. The European Food Safety Authority (EFSA) has established a list of allergens, including tree nuts, which must be declared in food according to Annex II of Regulation (EU) No 1169/2011.
3. Test Applications, Specificity, and Sensitivity
The AlerTox Stick Walnut test kit uses a monoclonal antibody combination specific for the major antigen of English/common walnut (i.e., the 11S globulin seed storage protein, also known as allergen Jug r 4, from Juglans regia). The kit is suitable for the following applications:
- Food samples
- Rinse water testing
- Surface testing
The limit of detection (LOD) for AlerTox Stick Walnut is 2.25 ppm walnut protein (2.25 mg of walnut protein per kg or liter of sample). The range of detection (ROD) is 2.25 - 206,000 ppm walnut protein (mg/kg or mg/L). Overload (signal decrease) may occur at concentrations between 10,000 - 206,000 ppm; however, hook effect (false negative) will not occur in this range.
For dry surfaces sampled with a wet swab, the LOD is approximately 4 micrograms of walnut protein per 16 cm² (2.5 in²). Refer to the Certificate of Analysis for LOD (search by lot number at: www.hygiena.com/documents).
Section 12 lists matrices for which the kit has been validated, with an LOD of 2.25 ppm walnut protein.
4. Kit Contents
| Component | KIT3092 |
|---|---|
| Individually foil-pouched walnut immunoassay test strips | 10 |
| Sample collection tubes with yellow caps | 10 |
| Sample extraction buffer in tubes with blue caps, 10 mL | 10 |
| Spoons | 10 |
| Transfer pipettes | 10 |
| 3 mL pipettes (for liquid sample testing only) | 10 |
| Swabs (for surface testing only) | 10 |
5. Other Materials Not Provided
- Grinder, mortar and pestle, or any other manual or automated homogenization system for sample pulverization
- Vortex mixer/shaker (recommended, not required)
- Scissors (for surface sampling only)
- Optional digital scale (0.1 g sensitivity)
6. Precautions
- All components in the kit should be stored at temperatures between 10 to 30 °C (50 to 86 °F).
- After opening the foil pouch, use the test strip within 10 minutes.
- Do not touch the white end of the test strip.
- Do not use if the test strip is damaged or the pouch is torn.
- Do not use the test strip after the expiration date.
- All kit components are for single use only and should not be reused.
- Do not mix components from different test kits.
7. Sample Handling
All samples must be brought to room temperature between 18 to 35 °C (64.4 to 95 °F) before use.
This test kit can detect target antigens in the following matrices:
- Solid food
- Liquid samples (e.g., beverages, rinse water from kitchens, cleaning water from cutting machines)
- Environmental surfaces
8. Test Procedure for Solid Food Samples
8.1 Before adding the sample to the yellow-capped tube, mash or crush it to obtain the finest possible debris. If possible, use a mortar and pestle or grinder.
8.2 Add 1 gram of sample to the yellow-capped tube. Alternatively, use the provided disposable spoon to add an equivalent amount of sample as per the table below.
| Food Type | Sample | Spoon |
|---|---|---|
| Flour, powders | Corn flour, rice flour, milk powder, spices | 2 |
| Crumbed products | Bread, biscuits, cakes, pastries | 2 |
| Meat, fish, and cured meats | Meat, fish, sausages, black pudding, pâté, canned meat and fish | 1 |
8.3 Pour the entire contents of the blue-capped tube (10 mL) into the yellow-capped tube.
Important: Retain the blue cap for later use.
8.4 Cap the yellow-capped tube and shake the sample using a vortex mixer for at least 20 seconds to ensure sample homogenization. Alternatively, shake the tube vigorously by hand.
8.5 Let stand for 2 minutes to allow solids to settle.
8.6 Use a transfer pipette to transfer the supernatant into the blue-capped tube. Note: For samples with high fat content, avoid the fatty layer in the supernatant.
8.7 Open the foil pouch, hold the test strip by the blue end, and carefully remove it. Place the white end of the test strip into the blue-capped tube. Note: Do not touch the white end of the test strip.
8.8 Wait 10 minutes to read the results. Note: Do not read the results after more than 10 minutes, as results may vary. Do not touch the test strip during this time.
Diagram showing the workflow for solid food samples: 1. Add sample to tube. 2. Add buffer. 3. Mix. 4. Let settle. 5. Transfer supernatant. 6. Insert test strip. 7. Read results (Positive/Negative) after 10 minutes.
9. Test Procedure for Liquid Samples
Liquid samples – beverages, rinse water from kitchenware, technical surfaces, or cutting machines – can be tested directly. Turbid samples should be filtered (using a paper or textile filter) or allowed to settle.
9.1 Using the provided 3 mL pipette, add 3 mL of liquid sample to the yellow-capped tube. If the sample is viscous (e.g., yogurt, sauce), use the provided disposable spoon to add an equivalent amount of sample to the yellow-capped tube as per the table below.
| Food Type | Example | Spoon |
|---|---|---|
| Liquids and sauces | Milk, fruit juice, condensed milk, yogurt, soup, gravy, sauces, butter, cream | 3 |
Note: Shake the sample to ensure it is homogeneous and that you are taking a representative test portion.
9.2 Add 3 mL of sample extraction buffer from the blue-capped tube to the sample in the yellow-capped tube.
Important: Retain the blue cap for later use.
9.3 Cap the yellow-capped tube and shake the sample using a vortex mixer for at least 20 seconds to ensure sample homogenization. Alternatively, shake the tube vigorously by hand.
9.4 If the liquid is turbid, let it stand for 2 minutes.
9.5 Use a transfer pipette to transfer the supernatant into the blue-capped tube. Note: For samples with high fat content, avoid the fatty layer in the supernatant.
9.6 Open the foil pouch, hold the test strip by the blue end, and carefully remove it. Place the white end of the test strip into the blue-capped tube. Note: Do not touch the white end of the test strip.
9.7 Wait 10 minutes to read the results. Note: Do not read the results after more than 10 minutes, as results may vary. Do not touch the test strip during this time.
Diagram showing the workflow for liquid samples: 1. Add liquid sample. 2. Add buffer. 3. Mix. 4. Let settle (if turbid). 5. Transfer supernatant. 6. Insert test strip. 7. Read results (Positive/Negative) after 10 minutes.
10. Test Procedure for Surface Analysis
Use a clean, unused swab to collect each sample. Swabs can be used for work surfaces or equipment.
10.1 Dip the swab into the buffer in the blue-capped tube to moisten it. Then, rub the surface in a zigzag pattern (at least 16 cm² / 2.5 in² or 40 cm² / 15.6 in²) with firm pressure, rotating the swab.
Note: Where possible, swab an area of approximately 4 cm x 4 cm (1.6 in x 1.6 in). For irregular surfaces, ensure consistent swabbing technique for each test. The area selected for analysis must be representative of the entire area of interest.
10.2 Place the swab into the sample collection tube and trim with scissors.
Note: After capping, the swab should fit into the yellow-capped tube.
10.3 Pour the entire contents of the blue-capped tube (10 mL) into the yellow-capped tube. Then, press the swab tip against the inner wall of the yellow-capped tube to extract the sample into the buffer.
Important: Retain the blue cap for later use.
10.4 Cap the yellow-capped tube and shake the sample using a vortex mixer for at least 20 seconds to ensure sample homogenization. Alternatively, shake the tube vigorously by hand.
10.5 Use a transfer pipette to transfer the supernatant into the blue-capped tube.
10.6 Open the foil pouch, hold the test strip by the blue end, and carefully remove it. Place the white end of the test strip into the blue-capped tube. Note: Do not touch the white end of the test strip.
10.7 Wait 10 minutes to read the results. Note: Do not read the results after more than 10 minutes, as results may vary. Do not touch the test strip during this time.
Diagram showing the workflow for surface analysis: 1. Swab surface. 2. Place swab in collection tube and trim. 3. Add buffer. 4. Mix. 5. Transfer supernatant. 6. Insert test strip. 7. Read results (Positive/Negative) after 10 minutes.
11. Result Interpretation
Two colored lines appear: One line in the control region (C) and another line in the test region (T) indicate a "Positive" result. The intensity of the test line color may vary but is not necessarily proportional to the concentration of walnut antigen in the sample.
One clear colored line appears in the control region (C) only: This indicates a "Negative" result.
No colored line appears in the control region (C): The test is invalid. Check the following and re-test with a new test strip:
- Sample handling
- Test procedure
- Expiration date
- Storage conditions
For further assistance, please contact Hygiena at www.hygiena.com/support.
Important Note!
AlerTox Sticks is a qualitative test used for sample screening in internal quality control. It should not be used as the sole basis for product safety assessment under any circumstances. It cannot replace laboratory quantitative analysis tests.
12. Validation
AlerTox Sticks Walnut has been validated with the following matrices:
| Matrix | Matrices Verified | Matrices Verified |
|---|---|---|
| Baked goods (including bread, breadsticks, biscuits, cakes, muffins) | Cereal and cereal snacks | Protein bars |
| Cheese | Dairy desserts (including ice cream) | Yogurt |
| Chocolate and chocolate bars | Waffles |
Matrices should be validated prior to use with AlerTox Stick Walnut. For more information on matrix validation, please contact Hygiena at www.hygiena.com/support.
13. Disclaimer
Intended Use: Hygiena products are intended for R&D, quality assurance, and quality control under the supervision of personnel with technical qualifications. Information obtained from Hygiena products should only be used in conjunction with the user's routine quality assurance program. Hygiena products are not intended to be the sole basis for product safety assessment. Data obtained from Hygiena products should not be used for human diagnostic or therapeutic purposes. Please read the "Warranty and Limitation of Liability" (available at www.hygiena.com/terms-and-conditions) before using the product.
These products are made from high-quality raw materials. Except for the quality of these materials, no warranty, express or implied, is made regarding their suitability, except for measuring target antigen content when used strictly in accordance with these instructions.
Using the kit for any other purpose is outside its intended use. For matrices not previously validated, Hygiena cannot guarantee the suitability of the kit and the accuracy of the results obtained in these matrices. Customers may choose to use the product on unvalidated food or surface matrices; however, Hygiena strongly recommends that users conduct their own validation testing to confirm suitability for their specific application. Any loss resulting from the use of this product, including indirect or special losses or expenses, is limited to the replacement value of the kit.
For additional information or assistance with matrix validation, please contact Hygiena at www.hygiena.com/support. All Hygiena terms and conditions apply and can be found at: www.hygiena.com/terms-and-conditions.
14. Contact Information
For more information, please visit www.hygiena.com/contact. For technical support, please visit www.hygiena.com/support.
15. Revision History
- INS3027 REVB, September 2020
Includes more detailed information on English/common walnut and black walnut. Includes graphical workflow for liquid samples. - INS-KIT3092-001-REVA, July 2025
Updated ROD. Standardized branding, wording, certain graphical workflows, and document ID numbers.
Manufacturer Information
Manufacturer:
Hygiena Diagnóstica España S.L.
P.I. Parque Plata
Calle Cañada Real 31 - 35
41900, Camas (Sevilla), Spain
www.hygiena.com
Distributor Information:
海净纳(上海)商贸有限公司 (Hygiena (Shanghai) Trading Co., Ltd.)
地址:上海市杨浦区黄兴路2218号1202室(上海合生汇) (Room 1202, No. 2218 Huangxing Road, Yangpu District, Shanghai (Shanghai Lifecenter))
电话:021-65060292 (Tel: 021-65060292)
邮箱:contact@hygiena.com (Email: contact@hygiena.com)
微信公众号:hygienachina (WeChat Official Account: hygienachina)
