AlerTox® ELISA Milk Kit

1. Introduction

Milk is one of the “Big 9” food allergens requiring product labeling. It is the third leading cause of food-induced anaphylaxis after peanuts and tree nuts. Milk allergies affect 2-3% of infants and children worldwide and may persist into adulthood. Accurate and reliable detection of major milk proteins is crucial for consumer safety and compliance with food labeling regulations.

The primary milk allergens are casein and β-lactoglobulin (BLG). In cow's milk, protein constitutes 38% of the non-fat solid content, with casein making up about 80% and BLG 10-15%. Casein is heat-stable and found in various foods and wine. BLG is not heat-stable and can be destroyed during food processing. BLG is present in the milk of most mammals, including cows, sheep, and goats, but not humans.

Hygiena offers three AlerTox® ELISA Kits specific for casein, BLG, or both (Milk). The AlerTox ELISA Milk Kit is a versatile option for testing foods and rinse or clean-in-place (CIP) water for both casein and BLG. The AlerTox ELISA Casein Kit is designed for wine and other food samples, while the AlerTox ELISA β-Lactoglobulin Kit is suitable for foods not heated above 60 °C (140 °F).

Note: Read this manual carefully before starting the test. The test must be performed by thoroughly trained staff.

1.1 Test Sensitivity and Specificity

The AlerTox ELISA Milk Kit detects and quantifies milk proteins (casein and BLG) in various food products, including bread, cakes, pastries, cookies, instant soups, wine, sausages, and meat salads. It can also be used for clean-in-place (CIP)/rinse water samples. For samples heated above 70 °C (158 °F), the kit primarily detects casein, as heat-denatured BLG may not be recognized by the anti-BLG antibodies.

The Limit of Detection (LOD) is 0.05 ppm (mg of milk proteins per kg or L of sample), and the Limit of Quantification (LOQ) is also 0.05 ppm. The detection range is quantitative between 0.5 and 10 ppm. Refer to Section 6.2.1 for detailed specifications and Section 4 for results calculations.

The cross-reactivity with other food matrices is as follows:

Refer to Sections 6.2.2 (Recovery) and 6.2.3 (Non-Cross Reactivity) for additional data.

Important: Do not modify the protocol regarding timing, temperatures, plate washing, pipetting volumes, or buffer types/pH values, as this will invalidate the test system.

1.2 Sample Preparation

Important: Sample preparation for this kit differs from most other AlerTox ELISA Kits. Use the resulting extracts only with the AlerTox ELISA Milk assay. Refer to Section 6.1 for compatibility with other AlerTox ELISA Kits.

1.3 Test Principle

The AlerTox ELISA Milk Kit employs a quantitative sandwich ELISA principle, where antigen concentration is directly proportional to color intensity.

1. Primary antibodies against milk proteins are immobilized on a microtiter plate. Standards or samples containing milk proteins are added to the wells. After a 20-minute incubation at room temperature (15-25 °C), wells are washed to remove unbound material.
2. Peroxidase-conjugated secondary antibodies against milk proteins are added. Following a second 20-minute incubation, the plate is washed again.
3. Substrate Solution is added, and after a 20-minute incubation, a blue color develops in positive wells. Stop Solution is added to halt color development, turning the wells yellow. The color is measured photometrically at 450 nm (OD_{450 nm}).

2. Materials and Storage

2.1 Materials Supplied in the Kit

2.2 Storage Conditions and Stability

• Store all kit components at 2-8 °C (36-46 °F) in the dark. DO NOT FREEZE.
• Return all reagents to 2-8 °C (36-46 °F) immediately after use.
• Diluted Washing Solution (1X) is stable for 4 weeks when stored at 2-8 °C (36-46 °F).
• Important: If precipitants form in the 10X Washing Solution, warm it at 37 °C (99 °F) for 15 minutes before dilution. Do not use if precipitants do not redissolve.
• Diluted Extraction & Sample Dilution Buffer (1X) is stable for 1 week when stored at 2-8 °C (36-46 °F).
• Important: If crystals precipitate in the 5X Extraction & Sample Dilution Buffer, warm it at 37 °C (99 °F) for 15 minutes before use. Do not use if precipitants do not redissolve.
• Diluted Standards are stable for at least 24 hours when stored at 2-8 °C (36-46 °F).
• Sample Extracts are stable for at least 24 hours at 2-8 °C (36-46 °F) or longer when frozen.

2.3 Material Required but Not Provided

• AlerTox Polyphenol Additive (Product No. ASY3213) for samples with polyphenols and antioxidants.
• 15-30 mL containers for extractions.
• Multi-channel pipettor: 50–200 µL.
• ELISA Plate Reader with 450 nm filter (e.g., MCH3005).
• Sterile pipette tips.
• Centrifuge.
• Pipettors: 10–100 µL, 100–1,000 µL.
• Distilled water.
• Stomacher, Mill, Mortar, Blender, etc.
• Water bath, adjustable to 60 °C (140 °F).
• Vortex mixer.

Examples of foods rich in polyphenols, tannins, and antioxidants include chocolate, tea, coffee, wine, purple corn, corn fiber, soy, berries, and legumes (e.g., chickpeas, lentils).

2.4 Optional Materials/Equipment

• Homogenizer for sample extraction.
• Repeating pipettor to minimize assay drift.
• Recommended: ELISA plate washer system for reduced washing time and improved consistency.

AlerTox ELISA Kits have been validated on automated ELISA systems. For validation details, contact Hygiena at www.hygiena.com/support.

3. Test Procedure

3.1 Reagent Preparation

Prepare reagents immediately before use, only in the quantity needed for the samples plus 5 standards. Duplicate measurements are recommended for each sample and standard.

Important: All reagents must be at room temperature (15-25 °C, 59-77 °F) before use.

3.1.1 Extraction & Sample Dilution Buffer

Dilute the 5X Extraction & Sample Dilution Buffer 1:5 with distilled water to create the 1X solution.

Important: If needed, redissolve precipitants by warming the 5X buffer at 37 °C (99 °F) for 15 minutes before dilution. Do not use if precipitants do not redissolve.

Note: Amounts needed per sample/standard:

* 10 mL total (including the zero standard).

3.1.2 Standards

Dilute all standards, including the zero standard, 1:100 with 1X Extraction & Sample Dilution Buffer.

Notes:

• We recommend mixing 20 µL of standard with 1,980 µL of 1X Extraction & Sample Dilution Buffer.
• Standard curve concentrations refer to the diluted standards.

3.1.3 Washing Solution

Dilute the 10X Washing Solution 1:10 with distilled water to create the 1X solution.

Important: If needed, redissolve precipitants by warming the 10X Washing Solution at 37 °C (99 °F) for 15 minutes before dilution. Do not use if precipitants do not redissolve.

Note: Approximately 2.5 mL of 1X Washing Solution is needed per well.

3.1.4 ELISA Plate

Prepare the ELISA plate by opening the foil bag, removing the required number of strips, and placing them into a frame.

Notes:

• When opening the foil bag for the first time, be careful not to cut the ziplock off.
• Store unused wells in the sealed foil bag with the drying agent at 2-8 °C (36-46 °F).

3.2 Sample Preparation

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

1. Resuspend the sample in 1X Extraction & Sample Dilution Buffer based on sample type:
• For solid samples:
  1. Maximize sample homogeneity by finely pulverizing at least 5 g of sample using a mortar, impact mill, or similar device.
  2. Resuspend 0.5 g of the homogenized mixture in 10 mL of 1X Extraction & Sample Dilution Buffer.
• For other liquid samples:
  1. Add 0.5 mL of the liquid sample to 9.5 mL of 1X Extraction & Sample Dilution Buffer.
2. Mix well.
3. Incubate the mixture for 15 minutes in a preheated water bath at 60 °C (140 °F), shaking samples every 2 minutes for homogeneity.
4. Centrifuge the mixture for 10 minutes at 2,000 x g at room temperature (15-25 °C, 59-77 °F). If the supernatant is not fully separated, filter it.
5. Cool the sample extract (supernatant or filtrate) to room temperature (15-25 °C, 59-77 °F). For meat and sausage samples, dilute the sample extracts 1:5 with 1X Extraction & Sample Dilution Buffer.

3.2.1 Workflow Overview

AlerTox ELISA Milk Kit Only

1. Add 0.5 mL liquid sample to a tube OR 1a. Homogenize solid sample, 1b. Weigh 0.5 g sample in a tube.
2. Add Extraction & Sample Dilution Buffer (9.5 mL for liquids, 10 mL for solids) and mix well.
   *Add Extraction Additive to polyphenol-containing samples before adding buffer.
3. Incubate 15 min at 60 °C with mixing.
   *For clear liquid samples, proceed to step 5.
4. Centrifuge at 2,000 x g for 10 min at RT and, if needed, filter.
5. Cool sample to RT*.
   *For meat and sausage samples, dilute samples 1:5 in Extraction & Sample Dilution Buffer before use.

Important: Sample preparation for this kit is different from other AlerTox ELISA Kits.

3.3 ELISA Procedure

Important: Critical factors for ELISA accuracy are temperature, timing, and plate washing. Insufficient washing leads to poor precision and false results.

Note: For better reproducibility, use an automated plate washer for steps 3 and 6.

1. Add 100 µL of diluted standards or sample extracts in duplicate to the microtiter plate wells.
   Note: See Section 7 for Example Assay Layout. If testing many samples, pipette one set of standards before samples and the second set after samples, then use the average values for calculations.
2. Incubate for 20 minutes at room temperature (15-25 °C, 59-77 °F).
   Important: Do not shake the plate during incubation.
3. Wash plates three (3) times with 300 µL of 1X Washing Solution per well.
   Note: After automated or manual washing, invert plates and strike against dry paper towels to empty 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 shake the plate during incubation.
6. Wash plates five (5) times with 300 µL of 1X Washing Solution per well.
   Note: Follow the same procedure as step 3 for emptying wells.
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 shake the plate during incubation.
9. Stop the enzyme reaction by adding 100 µL of Stop Solution (0.5 M H₂SO₄) to each well.
10. Gently shake the plate by hand and wait for 1 minute.
    Note: Blue wells turn yellow in the presence of casein and BLG.
11. Measure results using an ELISA plate reader with a 450 nm filter (OD_{450 nm}), following the instrument manufacturer's instructions.
    Note: Measure color change within 30 minutes.
    Important: If sample results fall outside the standard curve range, do not extrapolate. Dilute the sample extract further with 1X Extraction & Sample Dilution Buffer and repeat the ELISA test in duplicate.

3.3.1 Workflow Overview

Diagram:

1. Add 100 µL of sample/standard to each well.
2. Incubate 20 min at RT.
3. Wash 3X with 300 µL of 1X Wash Solution.
4. Add 100 µL of Conjugate Solution.
5. Incubate 20 min at RT.
6. Wash 5X with 300 µL of 1X Wash Solution.
7. Add 100 µL of Substrate Solution.
8. Incubate 20 min in the dark at RT.
9. Add 100 µL of Stop Solution.
10. Mix & wait 1 min.
11. Read results using a plate reader (450 nm).

4. Results Calculations

Results measure the concentration of whole milk proteins (casein and BLG). Conversion factors are provided for calculating results for meat, sausage, CIP water, and various milk products.

Standards are prepared for direct determination of whole milk protein concentrations. Sample dilutions during extraction are factored into calculations. Additional dilutions (e.g., for high sample concentration) require adjustment. Use the AlerTox ELISA Calculation Worksheet available at www.hygiena.com/documents or follow the instructions below.

Important: Do not use the AlerTox ELISA Calculation Worksheet if the Zero Standard is defined as the Blank for B - B₀ calculation.

Calculations use the arithmetic mean.

1. Calculate the mean OD value (OD_{450 nm}) for each set of duplicate standards and samples.
2. Subtract the mean Zero Standard value from each mean OD value (OD - OD_{Standard 0} = B - B₀).
   Important: If the Zero Standard is defined as the Blank in the plate reader software for B - B₀ calculation, skip this step.
3. Create the standard curve by plotting adjusted OD values of standards 1-4 on the y-axis against milk protein concentration (ppm) on the x-axis.
4. For each sample, find the B - B₀ value on the y-axis and read the corresponding concentration on the x-axis of the standard curve.

Notes:

• When using the standard sample preparation procedure (Section 3.2), the result for foodstuff samples does not need to be multiplied by the dilution factor of 20.
• For meat and sausage samples, multiply results by 5 to account for the additional dilution in sample extraction (Step 5).
• For rinse or CIP water, divide the result by 4 to compensate for the extraction dilution factor. The sample concentration will be in mg/L.

To convert ppm of milk protein to ppm of a milk product, multiply by the appropriate conversion factor:

* Conversion factors were determined during kit validation.

Example Assay Data

Example of a Typical Standard Curve

[Graph showing Target Antigen concentration on the x-axis and OD_450nm (B-B₀) on the y-axis, with a curve fitting equation and R² value.]

5. General Precautions

• If skin contact occurs with toxic or irritating substances, rinse the affected area with plenty of water and seek medical attention if needed. Refer to the Safety Data Sheet (SDS) available at www.hygiena.com/SDS.
• The Substrate Solution contains TMB, which is highly toxic if inhaled, ingested, or contacts skin. Refer to the SDS.
• The Stop Solution contains H₂SO₄, which is corrosive. Refer to the SDS.
• Handle the test kit in accordance with Good Laboratory Practices (GLP).
• Do not use reagents beyond the expiration date.
• Handle all solutions with gloves.
• Avoid cross-contamination during sample extraction.
• Clean devices like blenders after each sample preparation.
• Use sterile pipette tips.
• Do not exchange reagent vial caps.
• Do not interchange reagents between kits of different lot numbers.
• Do not alter reagents, as this can cause inaccurate results.
• Equilibrate all reagents to room temperature (15-25 °C, 59-77 °F) before use.
• Do not use solutions if they appear cloudy or have precipitated. Exceptions are 10X Washing Solution and 5X Extraction & Sample Dilution Buffer, which may have crystalline precipitates that must be dissolved (see Section 2.2).
• Substrate Solution is light-sensitive; avoid direct light exposure and store in the dark.
• Use only distilled water for diluting concentrated buffers.
• Do not allow wells to dry completely.
• Avoid incubating microtiter plates on cold work benches.

6. Additional Information

6.1 Sample Extraction Compatibility

Individual samples must be extracted separately when using the following kits:

* The AlerTox ELISA Histamine Kit uses a competitive ELISA test, while other AlerTox ELISA Kits use sandwich ELISA tests.
† Cheese and other food samples (except wine) require separate extraction.

The following AlerTox ELISA kits share the same sample preparation protocol, allowing sample extracts to be tested in 16 different ELISA Assays:

* BLG = β-lactoglobulin
† Only wine extract is compatible. Cheese and other food extracts are not compatible.
† STI = Soy trypsin inhibitor

6.2 AlerTox ELISA Milk Kit

6.2.1 Summary of Specifications

* ppm = mg of milk proteins per L or kg of sample.

6.2.2 Recovery

* Tested in typical matrices.

6.2.3 Non-Cross Reactivity

The following matrices were tested and found to be non-cross-reactive with the AlerTox ELISA Milk Kit:

Non-Cross-Reactive Matrices:

Adzuki bean, Almond, Apricot, Barley, Bean, white, Beef, Brazil nut, Buckwheat, Cabbage, white, Caraway, Cardamom, Carob gum, Carrot, Cashew, Cayenne, Celery, Cherry, Chestnut, Chia, Chicken, Chickpea, Chili, Cinnamon, Clove, Cocoa, Coconut, Cod, Corn, Cumin, Dill, Duck, Egg (dried), Fennel, Fenugreek, Flaxseed, Garden cress, Garlic (fresh), Garlic (granulated), Gelatin, cow, Ginger (fresh), Ginger (ground), Gliadin, Guar gum, Gum arabic, Hazelnut, Horseradish, Kidney bean, Kiwi, Lamb, Leek, Lentil, Lupine, Macadamia, Mustard, yellow, Nutmeg, Oats, Onion, Paprika, Pea, Peach, Peanut, Pecan, Pepper, black, Pine seed, Pistachio, Poppy seed, Pork, Potato, Prawn, Pumpkin seed, Radish, Rapeseed, Rice, Rye, Saccharose, Sesame, Shrimps, Soy flour, Soy lecithin, Split pea, Sunflower seed, Thyme, Tomato, Turkey, Turmeric, Walnut, Wheat.

7. Example Assay Layout

S0: Zero Standard (without antigen): mean value = B₀.
S1-S4: Standards: mean value = B.
SP: Samples: mean value = B.

8. Disclaimer

Field of Use: This Hygiena product is intended for research and development, quality assurance, and quality control under the supervision of technically qualified personnel. The generated information should be used in conjunction with the user's regular quality assurance program and not as the sole basis for assessing product safety for consumer release. Data obtained must not be used for human diagnostic or treatment purposes.

Before use, read the Limitation of Warranty and Liability available at www.hygiena.com/terms-and-conditions.

These products are manufactured from high-quality raw materials. Hygiena provides no warranty, expressed or implied, regarding suitability beyond measuring the target antigen content when used strictly according to these instructions, except for the quality of the materials themselves.

Using the kit for any other purpose is outside its intended scope. For unvalidated matrices, Hygiena cannot guarantee fitness for purpose or accuracy of results. Users may choose to test unvalidated matrices, but Hygiena strongly recommends performing their own fit-for-use testing. Hygiena's liability for damages arising from product use is limited to the replacement value of the kit.

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.

9. Contact Information

For more information, visit www.hygiena.com/contact. For technical support, visit www.hygiena.com/support.

10. Change Index

INS3022 REVD, July 2020: Clarified parts of the conversion factors table.

INS-KIT3041-001-REVA, June 2025: Updated recovery data, selectivity data, and document ID number. Included use of the AlerTox Polyphenol Additive for some sample preparations.

Appendix A. Specialized Sample Extraction Procedures

A.1 For Foods and Drinks Containing Polyphenols, Tannins or Antioxidants

Use this sample preparation protocol for foods and drinks rich in polyphenols, including tannins and antioxidants.

Representative Matrices: Berries, Chocolate, Corn fiber, Coffee, Corn, purple, Soy, Tea, Legumes (e.g., chickpeas, lentils), Wine.

Important: This procedure is not for use with the following kits:

• AlerTox ELISA Crustacean Kit
• AlerTox ELISA Histamine Kit
• AlerTox ELISA Lysozyme Kit
• Wine extracts for: AlerTox ELISA Casein Kit, AlerTox ELISA Ovalbumin Kit.

Procedure:

1. For solid samples: Maximize sample homogeneity by finely pulverizing at least 5 g of sample using a mortar, impact mill, or similar device.
2. For liquid samples: Proceed to Step b.
3. Mix the sample with the AlerTox Polyphenol Additive (Product No. ASY3213) and 1X Extraction & Sample Dilution Buffer, based on the kit used:
• i. For AlerTox ELISA Kits (except Hazelnut and Pistachio): Mix the sample and AlerTox Polyphenol Additive first, then add 1X Extraction & Sample Dilution Buffer (see table below) and mix well.
• ii. For AlerTox ELISA Hazelnut and Pistachio Kits: Dissolve 1 g of AlerTox Polyphenol Additive in 100 mL of 1X Extraction & Sample Dilution Buffer before mixing with the specified amount of sample (see table below).

* i.e., all AlerTox ELISA Kits except those specific for hazelnut, pistachio, milk or those excluded in the Important note above.

3. Incubate for 15 minutes in a preheated water bath at 60 °C (140 °F), shaking samples every 2 minutes to ensure homogeneity.
4. Centrifuge for 10 minutes at ≥ 2,500 x g.
5. If the supernatant is still not completely separated from the particulates, filter the supernatant.
6. Proceed with the ELISA Procedure (Section 3.3).

Important: Results calculations will not require additional dilution-factor adjustments for this procedure.

A.2 For Rinse Water or Clean-in-Place Water (AlerTox ELISA Milk Kit)

1. Adjust the pH of the sample to 9.5 ± 0.5.
2. Dilute 1 mL of the sample in 4 mL of 1X Extraction and Sample Dilution Buffer.
3. Proceed with the ELISA Procedure (Section 3.3).