AlerTox® ELISA Pistachio Kit Instructions

1.1 Test Sensitivity and Specificity

The AlerTox® ELISA Pistachio kit detects and quantifies pistachio proteins in various food products, including cakes, chocolate, desserts, pastries, pralines, puddings, and yogurts. It can also be used for rinse water or clean-in-place (CIP) samples. The limit of detection (LOD) is 0.07 ppm (mg of whole pistachio per kg or L of sample), and the limit of quantification (LOQ) is 1.0 ppm (mg of whole pistachio per kg or L of sample). The detection range is quantitative between 1.0 and 25 ppm. Refer to Section 6.2.1 for detailed specifications and Section 4 for result calculations.

Cross-reactivity with other food matrices is indicated in the following table:

Note: Almonds, chia, hazelnuts, pecans, radishes, Brazil nuts, chickpeas, leeks, poppy seeds, split peas, white cabbage, fenugreek, macadamia, pumpkin seeds, and walnuts showed results between 0.5 LOD and 1 LOD and may yield values above the LOQ.

Refer to Section 6.2.2 for Recovery and Section 6.2.3 for Cross-reactivity for additional data.

Important: Do not modify the protocol in terms of time, temperatures, plate washing, pipetting volumes, buffer types, or buffer pH. Any modification will invalidate the assay system.

1.2 Sample Preparation

The AlerTox® ELISA Pistachio kit is part of a series of twenty Hygiena® allergen test kits. Sixteen different allergens, including pistachios, can be detected using a single sample extract with these ELISA tests. Some allergens require individual extraction. Refer to Section 6.1 for sample extraction compatibility.

1.3 Test Principle

The AlerTox® ELISA Pistachio kit operates on the principle of a quantitative sandwich ELISA. The antigen concentration is directly proportional to the color intensity of the tested sample. The sandwich ELISA test involves the following steps:

1. Primary antibodies against pistachio proteins are immobilized on the surface of a microtitration plate. Standards or test samples containing pistachios are added to the wells. After a 20-minute incubation at room temperature (15-25°C / 59-77°F), unbound material is washed away.
2. Secondary antibodies conjugated to peroxidase and directed against pistachio proteins are added to the wells. After another 20-minute incubation, the plate is washed again.
3. Substrate solution is added, and the plate is incubated for an additional 20 minutes, resulting in a blue color development in positive wells. A stop solution is added to halt color development, turning the wells yellow. The yellow color is measured photometrically at 450 nm (OD450 nm).

2.1 Materials Provided in the Kit

2.2 Storage Conditions and Stability

• All kit components should be stored protected from light at a temperature between 2 and 8°C (36 and 46°F). DO NOT FREEZE.
• Return all reagents to 2-8°C (36-46°F) immediately after use.
• The diluted wash solution (1X) can be used for 4 weeks if stored at 2-8°C (36-46°F).
• Important: If necessary, redissolve precipitates by warming the 10X wash solution to 37°C (99°F) for 15 minutes before dilution. Do not use the buffer if the precipitate does not redissolve.
• The diluted extraction and sample diluent buffer (1X) can be used for one week if stored at 2-8°C (36-46°F).
• Important: If necessary, redissolve precipitates by warming the 10X extraction and sample diluent buffer to 37°C (99°F) for 15 minutes before dilution. Do not use the buffer if the precipitate does not redissolve.
• Sample extracts are stable for at least 24 hours at 2-8°C (36-46°F) or longer if frozen.

2.3 Required Materials Not Provided

• AlerTox Polyphenol Additive (Product No. ASY3213), for samples containing polyphenols and antioxidants*.
• Multichannel pipette: 50 - 200 µL
• Sterile pipette tips
• Pipettes: 10 - 100 µL, 100 - 1 000 µL
• Water bath, adjustable to 60°C (140°F)
• 15 to 30 mL tubes for extractions
• ELISA plate reader with a 450 nm filter (Absorbance 96 ELISA Reader, Product No. MCH3005, or similar)
• Centrifuge
• Distilled water
• Grinder, mill, mortar, blender, etc.
• Vortex mixer

* Chocolate, tea, coffee, wine, purple corn, and corn fiber, soy, berries, and legumes such as chickpeas or lentils are examples of foods rich in polyphenols, including tannins, and antioxidants.

2.4 Optional Materials/Equipment

• Homogenizer for sample extraction
• Repeating pipette to minimize assay drift
• Recommended: ELISA plate washer to reduce washing time and improve consistency.

AlerTox ELISA kits have been validated on fully automated ELISA systems (e.g., the BEAR automated ELISA robot). For validation details, contact Hygiena at www.hygiena.com/support.

3.1 Reagent Preparation

It is recommended to prepare reagents immediately before use, preparing only the quantity needed for the number of samples plus the 5 standards. Perform duplicate measurements for each sample and standard, following Good Laboratory Practices (GLP) and quality control requirements.

Important: All reagents must be at room temperature (15-25°C / 59-77°F) at the time of use.

3.2 Sample Preparation

Important: Refer to Appendix A for the sample preparation protocol for samples containing polyphenols, tannins, or antioxidants, as well as for rinse water or CIP. For other samples, follow the procedure below:

1. Resuspend the sample in the 1X Extraction and Sample Diluent Buffer according to the sample type:

1. Maximize sample homogeneity by finely grinding a minimum of 5 g of sample in a mortar, impact grinder, or similar device.
2. Resuspend 0.5 g of the homogenized mixture in 10 mL of 1X Extraction and Sample Diluent Buffer.

1. Add 0.5 mL of the liquid sample to 9.5 mL of 1X Extraction and Sample Diluent Buffer.

3.3 ELISA Procedure

Important: The most critical aspects of the ELISA procedure are temperature, time, and plate washing. Insufficient washing leads to inaccurate results.

Note: For better reproducibility, use of an automated, well-maintained plate washer is recommended for steps 3 and 6.

1. Add 100 µL of standards or sample extracts in duplicate to the appropriate wells of the microtitration plate.
2. Incubate for 20 minutes at room temperature (15-25°C / 59-77°F). Important: Do not agitate the plate during this incubation.
3. Wash the plates three (3) times with 300 µL of 1X wash solution per well. Note: At the end of automated washing or between manual washes, invert the plates and tap them against clean, dry paper towels to empty the wells and remove residual liquid.
4. Add 100 µL of conjugate solution to each well.
5. Incubate for 20 minutes at room temperature (15-25°C / 59-77°F). Important: Do not agitate the plate during this incubation.
6. Wash the plates five (5) times with 300 µL of 1X wash solution per well. Note: At the end of automated washing or between manual washes, invert the plates and tap them against clean, dry paper towels to empty the wells and remove residual liquid.
7. Pipette 100 µL of substrate solution into each well.
8. Allow the reaction to develop in the dark (substrate is light-sensitive) for 20 minutes at room temperature (15-25°C / 59-77°F). Important: Do not agitate the plate during this incubation.
9. Stop the enzymatic reaction by adding 100 µL of stop solution (0.5 M H₂SO₄) to each well.
10. Gently agitate the plate by hand and wait for 1 minute. Note: Wells with blue color will turn yellow in the presence of pistachio protein.
11. To measure the results, use an ELISA plate reader with a 450 nm filter (OD₄₅₀ nm), following the instrument manufacturer's instructions. Note: Measure color change within 30 minutes.

Important: If sample results fall outside the pistachio standard curve range, do not extrapolate data. Instead, further dilute the sample extract with 1X Extraction and Sample Diluent Buffer and repeat the ELISA test using the diluted sample extract and standards, in duplicate.

4. Results Calculation

Results are measured as concentrations of whole pistachios, not pistachio proteins. Refer to steps 4 and 5 below for conversion factors to calculate results for rinse water/CIP or pistachio protein concentrations, respectively.

Standards are prepared for direct determination of whole pistachio concentrations in samples. Sample dilution during extraction (as described in sample preparation procedures) is already accounted for in the level calculations. However, results should account for any additional dilution (e.g., due to high sample concentration or other extraction procedures) (Step 4, notes below). Use the AlerTox ELISA calculation spreadsheet (available at www.hygiena.com/documents) or the following instructions to calculate results.

Important: Do not use the AlerTox ELISA calculation spreadsheet if the plate reader software's zero standard is defined as the blank for the B - Bo calculation.

For result interpretation, the arithmetic mean is used for calculations.

1. Calculate the average optical density (OD₄₅₀ nm) value for each set of duplicate standards and duplicate samples.
2. Subtract the average zero standard value from each average OD value of standards or samples (OD - ODStandard 0 = B - Bo). See below for Example Assay Data. Important: If the plate reader software's zero standard is defined as the blank for the B - Bo calculation, skip this step.
3. To create the standard curve, plot the adjusted optical density values of standards 1 through 4 on the y-axis against the whole pistachio concentration in ppm on the x-axis. See below for a typical standard curve example.
4. For each sample extract, find the B - Bo value on the y-axis. Then, read the corresponding value on the x-axis of the standard curve to determine the whole pistachio concentration.

Remarks:

• When using the standard sample preparation procedure (Section 3.2), it is not necessary to multiply the resulting food sample concentration by the 20-fold dilution factor.
• When analyzing rinse water or CIP, divide the result by 4 to compensate for the dilution factor used in that extraction procedure. (The sample concentration will be expressed in mg/L.)

5. To convert ppm of whole pistachios to ppm of pistachio protein, divide the results by 4.9 [1].

5. General Precautions

• If your skin comes into contact with toxic or irritating substances, rinse the affected area with plenty of water and consult a doctor if necessary. Consult the SDS, available at: www.hygiena.com/SDS.
• The substrate solution contains TMB, which is highly toxic if inhaled, ingested, or in contact with skin. Refer to the SDS.
• The stop solution contains H₂SO₄, which is corrosive. Refer to the SDS.
• Handle the test kit according to GLP.
• Do not use reagents beyond the kit's expiration date.
• Handle all solutions with gloves.
• During sample extraction, avoid cross-contamination.
• Equipment, such as mixers, should be cleaned after each sample preparation.
• Use sterile pipette tips.
• Do not swap reagent vial caps.
• Do not interchange reagents between kits with different lot numbers.
• Do not modify reagents, as this may lead to inaccurate results.
• All reagents should be equilibrated to room temperature (15-25°C / 59-77°F) before use.
• Do not use solutions if they become cloudy or precipitate. Exceptions are the 10X wash solution and 10X extraction and sample diluent buffer, which may contain crystalline precipitates that must be completely dissolved before use (see Section 2.2).
• The substrate solution is light-sensitive. Avoid exposure to direct light and store in the dark.
• Use only distilled water to dilute concentrated buffers.
• Do not allow wells to dry out completely.
• Avoid incubating microtitration plates on cold surfaces.

6. Additional Information