Enzyme-linked immunoassay (ELISA) for total aflatoxin

Enzyme-linked immunoassay (ELISA) for total aflatoxin

Kit instruction manual

This kit is for research use only

1 Purpose: This kit is used for quantitative detection of total aflatoxin residues in feed, fish, shrimp and meat tissues (eg chicken, beef and pork), eggs, honey, milk, serum and urine samples.

2 Experimental principle

This kit uses a competitive ELISA method. The microplate is coated with the total aflatoxin conjugated antigen. The total aflatoxin standard or sample is added. The free total aflatoxin and the pre-coated total aflatoxin on the microwell strip Toxin-conjugated antigens compete with each other for anti-total aflatoxin antibody enzyme markers, which are developed with TMB substrate. The color changes from blue to yellow after addition of the stop solution. The microplate reader is used to detect at 450nm wavelength. The total aflatoxin content is inversely proportional to the standard curve to calculate the total aflatoxin content in the sample.

3 Kit composition

3.1 Removable enzyme labeling plate of total aflatoxin pre-coated antigen: 1 piece (12 wells × 8 strips).
3.2 Total aflatoxin standard: 6 bottles (1ml / bottle), the contents are: 0 ng / ml, 0.5 ng / ml, 2 ng / ml, 20 ng / ml, 200 ng / ml, 400 ng / ml
3.3 Anti-total aflatoxin antibody enzyme conjugate: 1 bottle (6ml).
3.4 Color Rendering Solution A: 1 bottle (6ml).
3.5 Color Rendering Solution B: 1 bottle (6ml).
3.6 Stop solution: 1 bottle (6ml), 2M sulfuric acid.
3.7 Sample diluent: 1 bottle (10 ×, 6ml), used for sample dilution.
3.8 Concentrated washing solution: 1 bottle (20 ×, 20ml), used for washing plates.
3.9 A manual.

4 Materials needed but not provided
4.1 Equipment
4.1.1 Wavelength 450nm microplate reader.
4.1.2 Crusher.
4.1.3 Measuring cylinder.
4.1.4 Oscillator.
4.1.5 Funnel.
4.1.6 Whatman No 1 or equivalent filter paper.
4.1.7 Micro pipette.
4.2 Reagent
4.2.1 Deionized water or distilled water.
4.2.2 Methanol.
5 Storage
5.1 The kit is stored at 2 ~ 8 ℃, do not freeze
5.2 Unused microplates should be sealed and dried
6 Notes
6.1 Please read the instructions carefully before using the kit.
6.2 Do not use expired kits.
6.3 Before using the kit, return the reagent to room temperature (25 ± 2 ℃). It is recommended to return to temperature for at least 2 hours.
6.4 The standard contains total aflatoxin. Special care should be taken when using it. Gloves should be worn during operation.
6.5 The stop solution contains sulfuric acid, which prevents burns to the skin and corrosion of clothing when used.
6.6 The tips used for different standards and samples can not be mixed, otherwise it will affect the test results.
6.7 The reagents in different batch kits should not be mixed; the tips used for different standards and samples should not be mixed, otherwise it will affect the experimental results.
6.8 When diluting the sample, the sample diluent in this kit must be used, otherwise it will affect the experimental results
6.9 Avoid foaming when mixing reagents.
7 Working fluid preparation
7.1 Total aflatoxin standard solution: 0 ng / ml, 0.5 ng / ml, 2 ng / ml, 20ng / ml, 200 ng / ml, 400 ng / ml
7.2 Concentrated washing solution: dilute with distilled water at 1:20 (1 + 19) for later use

7.3 Sample diluent: spare
7.3 Developer: reserved, avoid direct light
7.4 Reaction stop solution: reserved
8 Sample processing procedures (samples should be strictly operated in accordance with the instructions during the extraction process, and they should be accurately diluted during the extraction process, otherwise the results will be inaccurate, and the samples should be stored in a cool and dark place and stored in refrigerator)
8.1 Take 10g crushed sample and add 20ml 70% methanol solution
8.2 Vibrate vigorously for 3 minutes
8.3 Filter with Whatman No 1 filter paper
8.4 Take 25µl of the processed sample and add 25µl of sample diluent to the reaction well (sample dilution factor is 2)
9 Enzyme-free analysis steps
9.1 Experimental notes
9.1.1 Before starting the experiment, please restore all reagents to room temperature (25 ± 2 ℃) outside the box for about 2 hours. After warming to room temperature (25 ± 2 ℃), take out the microporous strips again. Re-seal the microporous strips and dry immediately at 2 ~ 8 ℃. Note: Make sure that the temperature is sufficient, otherwise the accuracy and accuracy of the test will be affected.
9.1.2 Please put the reagent back to 2 ~ 8 ℃ immediately after use
9.1.3 Please do not change the analysis program
9.1.4 Please use an accurate micropipette
9.1.5 Once the operation starts, please do not interrupt any program
9.1.6 The reproducibility of ELISA results greatly depends on the operating procedures, please strictly follow the requirements
9.1.7 To avoid cross-contamination, each standard and sample should be loaded with different tips
9.1.8 Do not let the tip touch the solution or inner surface of the microwell when loading
9.2 Analysis steps
9.2.1 Number in advance, mark the position of B0, standard and sample, double hole detection is recommended
9.2.2 Take the required number of micropores (micropore strips are removable), reseal the excess strips and immediately put them back at 2 ~ 8 ℃ to store
9.2.3 The sample diluent (10 ×) and concentrated washing solution (10 ×) are diluted into working solution (diluted with distilled or deionized water)
9.2.4 Add 50µl of 0.0 ng / ml standard solution to well B0
9.2.5 Add 50 μl of standard solution to each standard well
9.2.6 Add 50µl of sample solution to each sample well
9.2.7 Add 50µl of anti-total aflatoxin antibody enzyme conjugate to all wells
9.2.8 Gently shake the reaction plate for a few seconds.
9.3 Warm bath at 37 ° C for 30min (Tap the reaction plate from time to time during the warm bath to reduce double-hole errors)
9.3.1 Shake off the liquid in the well and wash the microplate 5 times with lotion. The last time should be tapped on absorbent paper to completely remove the liquid in the well.
9.4 Response
9.4.1 After the washing procedure is completed, immediately add 50µl of chromogenic solution A and then 50µl of chromogenic solution B to each microwell with a micropipette; shake the reaction plate slightly to mix thoroughly
9.4.2 37 ℃ warm bath for 10min
9.4.3 Add 50µl of stop solution to each well and mix well
9.4.4 Detect the absorbance at 450nm and read the result within 5min.
10 Result calculation
10.1 Quantitative analysis
10.1.1 The average value (B) of the absorbance value of each concentration standard solution and sample obtained is divided by the absorbance value (B0) of the first standard (0 standard) and multiplied by 100%, which is the percent absorbance value.
B—average absorbance value of standard solution or sample solution
Average absorbance value of B0—0µg / L standard solution
10.1.2 With the logarithmic value of the total aflatoxin concentration as the X axis and the percent absorbance value as the Y axis, draw a standard curve. According to the percent absorbance of the sample, the abscissa of the corresponding point can be obtained from the curve, which is the logarithmic value of the total aflatoxin concentration, and the antilogarithm is the total aflatoxin concentration C (ppb) in the measurement solution.
10.1.3 Because the sample has been pre-diluted, the sample concentration obtained from the standard curve must be multiplied by its dilution factor.
10.2 Semi-quantitative determination
10.1.1 Visual semi-quantitative determination: first select an appropriate standard solution to run with the sample, and judge whether the concentration value of the sample is less than or greater than the standard value based on the comparison of the absorbance value of the sample and the standard.
10.1.2 Instrument semi-quantitative determination: first select an appropriate standard solution to run with the sample, and determine whether the concentration value of the sample is less than or greater than the standard value according to the color depth comparison of the sample and the standard product.