2. Technical notes and limitations

• If room temperature is below 25 °C or reagents/samples have not been equilibrated to room temperature (20–25 °C), standard OD values may be lower than expected.
• If the microplate dries during washing, non-linear standard curves and poor reproducibility may occur. Therefore, proceed immediately to the next step after  washing.
• Mix all reagents thoroughly before adding them.
• Do not use the kit after its expiration date. The use of diluted or adulterated reagents will alter the sensitivity and OD values obtained. Do not mix reagents from different lots.
• Storage conditions: Keep at 2–8 °C, do not freeze. Unused microplates should be placed in a self-sealing bag to re-seal. The standard and the colorless substrates are light-sensitive; avoid direct exposure.
• Discard any coloring solution that shows color changes, which indicates deterioration. If the 0 ppb standard reading is less than 0.5 (A450 nm < 0.5), the reagent has deteriorated.
• The optimal reaction temperature is 25 °C; excessively high or low temperatures will affect sensitivity and OD values.
• A standard curve must be generated for each batch of samples analyzed.
• It is recommended to analyze all standards and samples in duplicate.

3. Precautions

• The stop solution suggested for use with this kit is an acidic solution. Wear protective gloves, clothing, and eye and face protection. Wash your hands thoroughly after handling.

4. Technical specifications

• Sensitivity: 0.03 ppb
• Incubation temperature: 25 °C
• Incubation time: 30 min ~ 15 min
• Limit of detection:
  • Milk: 0.1 ppb
  • Milk powder, Yogurt: 0.3 ppb
• Cross-reactivity rate:
  • Aflatoxin M1: 100%
  • Aflatoxin B1: 12.9%
  • Aflatoxin B2: 1.4%
  • Aflatoxin G1: 1.9%
  • Aflatoxin G2: 0.3%
• ​ Recovery rate:
  • Milk: 90 ± 25%
  • Milk powder, Yogurt: 100 ± 30%

5. Components

6. Materials required but not included

1. Equipment: ELISA reader (450 nm/630 nm), homogenizer, shaker, centrifuge,
   balance: sensitive to 0.01 g, nitrogen drying device, incubator, graduated pipettes, printer.
2. Micropipettes: single-channel 20 μL – 200 μL, 100 μL – 1000 μL, multichannel 30 – 300 μL.

7. Sample pretreatment

Instructions (The following points must be addressed before pretreatment):

• Only disposable tips should be used and must be changed each time they are used to aspirate different reagents.
• Before the experiment, each experimental utensil must be clean and, if necessary,
  cleaned again to avoid contamination that interferes with the results.

Preparation of solutions before sample pretreatment:

• Sample redissolution solution: Use 1 part of the 10× concentrated redissolution solution and mix with 9 parts of deionized water to obtain the ready-to-use solution.

Sample preparation

7.1 Preparation of raw milk and processed milk samples

1. Take raw or processed milk samples, keep them at room temperature, and
   analyze them directly when they reach that temperature.
2. Take 50 μL for the assay. (Dilution factor: 1).

7.2 Preparation of milk powder samples

1. Place 1.0 ± 0.05 g of milk powder sample in a 50 mL centrifuge tube; add 10 mL
   of sample redissolution solution, shake thoroughly for 3 minutes, and centrifuge at more than 4000 rpm at 20 °C for 10 minutes.
2. Take 50 μL of the supernatant for the assay (Dilution factor: 10).

7.3 Preparation of yogurt samples

1. Take 1 mL of yogurt sample and dilute with sample redissolution solution at a
   ratio of 1:9 (100 μL of yogurt + 900 μL of redissolution solution). Mix for 30
   seconds.
2. Take 50 μL for the assay (Dilution factor: 10).

8. ELISA procedure

8.1 Instructions

1. Bring ELISA reagents to room temperature (20–25 °C) before use.
2. After use, return reagents immediately to 2–8 °C.
3. The reproducibility of the ELISA analysis largely depends on consistent plate washing; correct washing operation is key to assay accuracy.
4. Throughout the constant-temperature incubation process, avoid light exposure
   and seal the microplate with the protective membrane.

8.2 Operating procedures

1. 8.1 Instructions
2. Bring ELISA reagents to room temperature (20–25 °C) before use.
3. After use, return reagents immediately to 2–8 °C.
4. The reproducibility of the ELISA analysis largely depends on consistent plate
   washing; correct washing operation is key to assay accuracy.
5. Throughout the constant-temperature incubation process, avoid light exposure
   and seal the microplate with the protective membrane.
6. 8.2 Operating procedures
7. Bring the test kit to room temperature (20–25 °C) for at least 30 minutes; ensure
   that each reagent is well mixed before use.
8. Place the required microwell strips into the plate frames. Reseal unused
   microplates and store them at 2–8 °C (do not freeze).
9. Preparation of the wash solution: take 40 mL of 20× concentrated wash buffer
   and dilute with deionized water at a ratio of 1:19 (1 part buffer + 19 parts water),
   or prepare according to the required amount.
10. Numbering: number the wells according to the samples and the standard
    solutions; each sample and each standard must be analyzed in duplicate,
    recording their positions.
11. Addition of standard/sample: add 50 μL of the sample or standard solution to
    duplicate wells, then add 50 μL/well of enzyme conjugate and 50 μL/well of
    antibody working solution. Mix gently by manually shaking the plate, seal it with
    the membrane, and incubate at 25 °C for 30 minutes in the dark.
12. Microplate washing: Carefully open the protective membrane and pour out the
    liquid from the wells. Add 250 μL/well of wash buffer and wash thoroughly 4–5
    times, 15 to 30 seconds each time. Then remove the liquid and dry by gently
    tapping the plate on absorbent paper. (If bubbles remain after drying, use a clean
    tip to pierce them.)
13. Color development: Add 50 μL of Substrate Solution A, then 50 μL of Substrate
    Solution B to each well. Mix gently by manually shaking the plate and incubate at 25 °C for 15 minutes in the dark for color development.
14. ​Reading / Determination: Add 50 μL of Stop Solution to each well. Mix gently by manually shaking the plate. Set the wavelength of the microplate reader to 450 nm to determine the OD (optical density) value of each well. (It is recommended to measure the OD value at dual wavelength: 450/630 nm, within 5 minutes after adding the Stop Solution).

9. Interpretation of results

There are two methods to evaluate the results: the first is approximate evaluation, while the second is quantitative determination. The sample OD value has a negative correlation with the aflatoxin M1 present in the sample.

9.1 Qualitative determination

The concentration range (ppb) can be obtained by comparing the average absorbance value with the standards. Suppose the absorbance value of sample one is 0.3, sample two is 1.0, and the standards are: 0 ppb of 2.243; 0.03 ppb of 1.816; 0.09 ppb of 1.415; 0.27 ppb of 0.74; 0.81 ppb of 0.313; 2.43 ppb of 0.155. Then the concentration of sample one is in the range of 0.81 ppb ~ 2.43 ppb; sample two is in 0.09 ppb ~ 0.27 ppb. The concentration range of aflatoxin M1 in the samples can be obtained by multiplying by the corresponding sample dilution.

9.2 Quantitative analysis

To calculate the concentration of the samples, a standard curve must be constructed.
Before constructing it, the concept of % absorbance (%B/B0) should be understood.
B Percent absorbance = B × 100 % 0

The 0 standard is taken as 100%, and the absorbance values of the other standards
are expressed as relative percentages.

Then, the calculated values for the standards are plotted on a semi-logarithmic
coordinate system, where the X-axis represents the concentration of Aflatoxin M1
(μg/L or ppb) and the Y-axis represents % absorbance.

The concentration of Aflatoxin M1 (in μg/L or ppb) corresponding to the absorbance of
each sample can be determined from the calibration curve. Special software for
ELISA result analysis can facilitate double or multiple determinations.