AlerTox Sticks Mustard Seeds

1. Intended Use

The AlerTox® Sticks Mustard Seeds Kit is designed for the qualitative detection of mustard seed antigen in food, kitchens, and production facilities. Samples prepared according to these instructions can be tested using strips from various AlerTox Sticks kits, but only specific kits are compatible. Users must read all instructions before commencing the assay.

2. Introduction

Mustard is derived from the seeds of various mustard plants, including white, yellow, brown, Indian, and black mustard varieties. Oriental mustard and wasabi are also part of the mustard family. Mustard allergies can manifest with symptoms ranging from mild oral reactions to severe systemic responses like anaphylactic shock. While considered a major food allergen in some regions, its global prevalence is not precisely estimated. European Union regulations mandate allergen labeling, including mustard, while US regulations (FALCPA) do not currently list mustard as a major allergen.

3. Test Applications, Specificity and Sensitivity

This kit utilizes monoclonal antibodies targeting the legumin-like 11S seed storage protein (cruciferin, CRU4), also known as allergen Sin a 2. It detects antigens from all mustard varieties, including wasabi. Suitable applications include:

• Food samples
• Rinse water testing
• Surface testing

The Limit of Detection (LOD) is 2 ppm of raw, unprocessed mustard seed protein. The Range of Detection (ROD) is 2 – 237,500 ppm of hazelnut protein. Overloading may occur at higher concentrations, but a total hook effect (false negative) is not observed within this range. LODs for surface testing can be found on the Certificate of Analysis. AlerTox Sticks is a qualitative assay; for quantification, the AlerTox ELISA Mustard Kit (KIT3058) should be used.

Notes:

• High cross-reactivity is observed with other cruciferous family seeds like broccoli, Brussels sprouts, cabbage, cauliflower, horseradish, radish, rutabaga, and turnip.
• Non-reactive to rapeseed, canola oil, and edible parts of other cruciferous members.
• Sensitivity may decrease under specific conditions:
  • Samples heated above 150 °C (302 °F).
  • Fat-rich environments (e.g., presence of oil or creams).
  • Matrices with low pH (e.g., vinegar, citric acid).
  • Testing performed below 18 °C (64.4 °F).
• Viscous, dense, or high-fat samples may affect migration and assay results. Contact Hygiena for information on sample extraction and dilutions.

4. Kit Contents

5. Other Materials Not Supplied

• AlerTox Polyphenol Additive (Product No. ASY3213) for samples with polyphenols/antioxidants (e.g., 70% chocolate).
• Grinder, mortar, or homogenization system for sample preparation.
• Vortex mixer/shaker (recommended).
• Scissors (for surface sampling).
• Optional: Digital scale (0.1 g sensitivity).

Note: Foods rich in polyphenols and antioxidants include berries, corn fiber, purple corn, legumes, soy, tea, and wine. These require validation with the AlerTox Polyphenol Additive.

6. Precautions

• Store kit components at 10-30 °C (50-86 °F).
• Use test strips within 10 minutes of opening the foil pouch.
• Do not touch the white end of the test strip.
• Do not use broken, damaged, or expired test strips.
• All kit components are disposable; do not reuse.
• Do not combine components from different kits.

7. Sample Handling

All samples must be at 18-35 °C (64.4-95 °F) before use. The test is designed for:

• Solid food
• Liquid samples (beverages, wash water from cutting equipment)
• Surfaces

8. Test Procedure for Solid Food Samples

Important: For samples containing polyphenols or antioxidants (e.g., 70% chocolate, excluding coffee), add 1 spoonful of AlerTox Polyphenol Additive (ASY3213) to a yellow-capped tube before proceeding.

1. Sample Preparation: Mash or crush the sample into fine crumbs using a mortar or grinder.
2. Add Sample: Add 1g of the sample to a yellow-capped tube. Alternatively, use the provided spoons for equivalent amounts based on food type:

   Food Type	Examples	Spoonfuls
   Flours, fine powders	Corn flour, rice flour, milk powder, spices	2
   Fine crumbs	Bread, cookies, cakes, snacks	2
   Meat, fish and cured meat	Meat, fish, sausage, black pudding, pâté, canned meat and fish	1

3. Add Buffer: Pour the entire contents of a blue-capped tube (10 mL) into the yellow-capped tube. Keep the blue cap.
4. Homogenize: Close the yellow-capped tube and shake vigorously for at least 20 seconds using a vortex mixer or by hand.
5. Settle Solids: Let the tube rest for 2 minutes to allow solids to settle.
6. Collect Supernatant: Use a small pipette to fill the blue cap with the clear liquid (supernatant), avoiding any fat layer.
7. Insert Test Strip: Open the foil pouch, hold the BLUE end of the test strip, and place the white end into the blue cap. Do not touch the white end.
8. Read Result: Wait 10 minutes. Do not read results after more than 10 minutes.

Workflow Visual Description:

1. Sample preparation (crushing). 2. Adding sample to tube. 3. Adding buffer. 4. Shaking for 20 seconds. 5. Settling for 2 minutes. 6. Pipetting supernatant. 7. Inserting test strip. 8. Reading result after 10 minutes (Positive/Negative indicators shown).

9. Test Procedure for Liquid Samples

Liquid samples (beverages, wash water) can be tested directly. Turbid samples should be filtered or allowed to settle.

Important: For samples containing polyphenols or antioxidants (e.g., 70% chocolate, excluding coffee), add 1 spoonful of AlerTox Polyphenol Additive (ASY3213) to a yellow-capped tube before proceeding.

1. Add Sample: Using a 3 mL pipette, add 3 mL of liquid sample to a yellow-capped tube. For thick samples (yogurt, sauce), use spoons for equivalent amounts:

   Food Type	Examples	Spoonfuls
   Liquid and sauces	Milk, juice, condensed milk, yogurt, soup, gravy, sauce, cream	3

   Shake the sample to ensure homogeneity.
2. Add Buffer: Add 3 mL of sample extraction buffer from a blue-capped tube using the 3 mL pipette. Keep the blue cap.
3. Homogenize: Close the yellow-capped tube and shake vigorously for at least 20 seconds using a vortex mixer or by hand.
4. Settle (if cloudy): If the liquid is cloudy, let it settle for 2 minutes.
5. Collect Supernatant: Use a small pipette to fill the blue cap with the clear liquid (supernatant), avoiding any fat layer.
6. Insert Test Strip: Open the foil pouch, hold the BLUE end of the test strip, and place the white end into the blue cap. Do not touch the white end.
7. Read Result: Wait 10 minutes. Do not read results after more than 10 minutes.

Workflow Visual Description:

1. Adding liquid sample to tube. 2. Adding buffer. 3. Shaking for 20 seconds. 4. Settling for 2 minutes (if needed). 5. Pipetting supernatant. 6. Inserting test strip. 7. Reading result after 10 minutes (Positive/Negative indicators shown).

10. Test Procedure for Surface Analysis

Use a clean, unused swab to collect samples from working surfaces or equipment.

1. Wet Swab: Dip the swab into the blue-capped tube.
2. Swab Surface: Firmly rub and rotate the swab on the testing surface using a zigzag pattern (at least 16 cm² or a 40 cm line). For irregular surfaces, maintain consistent technique. The area should be representative of the total area of interest.
3. Trim Swab: Place the swab into the sample collection tube and trim it using scissors. Ensure it fits within the yellow-capped tube when closed.
4. Add Buffer: Pour the entire contents of a blue-capped tube (10 mL) into the yellow-capped tube. Press the swab tip against the inside wall to facilitate extraction into the buffer. Keep the blue cap.
5. Homogenize: Close the yellow-capped tube and shake vigorously for at least 20 seconds using a vortex mixer or by hand.
6. Collect Supernatant: Use a small pipette to fill the blue cap with the clear liquid (supernatant).
7. Insert Test Strip: Open the foil pouch, hold the BLUE end of the test strip, and place the white end into the blue cap. Do not touch the white end.
8. Read Result: Wait 10 minutes. Do not read results after more than 10 minutes.

Workflow Visual Description:

1. Wetting swab. 2. Swabbing surface. 3. Placing swab in tube and trimming. 4. Adding buffer. 5. Shaking for 20 seconds. 6. Pipetting supernatant. 7. Inserting test strip. 8. Reading result after 10 minutes (Positive/Negative indicators shown).

11. Interpretation of Results

POSITIVE: Two colored lines appear – one in the control zone (C) and one in the test zone (T). The test line intensity may vary.

NEGATIVE: Only one colored line is clearly visible in the control zone (C).

INVALID: If no colored line appears in the control zone (C), the test is invalid. Re-run the test after checking:

• Correct specimen handling.
• Correct test procedure.
• Expiration date.
• Correct storage conditions.

For assistance, contact Hygiena at www.hygiena.com/support.

IMPORTANT NOTE: AlerTox Sticks is a qualitative screening test for internal quality control and cannot replace laboratory analysis for quantification.

12. Validation

AlerTox Sticks Mustard Seeds has been validated for the following matrices:

* Pure oriental and ground yellow mustard may show decreased sensitivity.
† 70% chocolate samples were prepared using AlerTox Polyphenol Additive.
‡ Coffee samples, though containing polyphenols, were not prepared with the AlerTox Polyphenol Additive.

Matrices require validation before use. For matrix validation information, contact Hygiena at www.hygiena.com/support.

13. Disclaimer

This product is intended for research, development, quality assurance, and quality control by technically qualified persons. Information generated should complement, not replace, a user's regular quality assurance program. It should not be the sole basis for product safety assessment for consumers. Data must not be used for human diagnostic or treatment purposes. Refer to the Limitation of Warranty and Liability at www.hygiena.com/terms-and-conditions before use.

Hygiena warrants materials are high-quality but makes no other warranties regarding suitability beyond exact adherence to instructions. Use for purposes other than intended is not covered. Hygiena cannot guarantee accuracy for unvalidated matrices; users performing such testing should conduct their own fit-for-use assessments. Liability is limited to kit replacement value.

For additional information or assistance with matrix validation, 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, visit www.hygiena.com/contact.

For technical support, visit www.hygiena.com/support.

15. Change Index

• INS3021 REVB, April 2022: Initial instructions.
• INS-KIT3040-001-REVA, July 2025: Updated LOD and ROD information. Included use of AlerTox Polyphenol Additive for sample preparations. Standardized branding, wording, graphic workflows, and document ID.