Aflatoxin B1 (AFB1) enzyme-linked immunosorbent assay (ELISA) kit instruction manual

This kit is for research use only.

1 Purpose of use :

This kit is used for the quantitative detection of aflatoxin B1 (AFB1) residues in corn, rice, wheat, beans, peanuts and peanut butter.

2 Experimental principle

The kit uses a direct competition ELISA method, coated with aflatoxin B1 (AFB1) antigen in a microplate, aflatoxin B1 (AFB1) standard or sample, free aflatoxin B1 (AFB1) microporous The pre-coated aflatoxin B1 (AFB1)-conjugated antigen on the strip competes with each other against the aflatoxin B1 (AFB1) somatic enzyme marker, and the color is changed with the TMB substrate, and the color changes from blue to yellow after the addition of the stop solution. The absorbance was inversely proportional to the aflatoxin B1 (AFB1) content in the sample by a microplate reader. The content of aflatoxin B1 (AFB1) in the sample was calculated by a standard curve.

3 kit composition

3.1 Pre-coated aflatoxin B1 (AFB1) coupled antigen detachable plate: 1 block (12 holes × 8).
3.2 Aflatoxin B1 (AFB1) standard: 6 bottles (1ml / bottle), the content is: 0 ppb 0.2 ppb, 0.6 ppb, 1.8ppb, 5.4ppb, 16.2ppb.

3.3 Anti-Aflatoxin B1 (AFB1) Antibody Enzyme Conjugate: 1 bottle (6 ml).
3.4 Coloring solution A: 1 bottle (6 ml).
3.5 color developing solution B: 1 bottle (6 ml).
3.6 Stop solution: 1 bottle (6 ml), 2 M sulfuric acid.
3.7 Sample Diluent: 1 bottle (10×, 6 ml) for sample dilution.
3.8 Concentrated washing solution: 1 bottle (20 x, 20 ml) for washing the plate.
3.9 a copy of the instructions.

4 materials that are not available

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 Micropipettes.
4.2 Reagents
4.2.1 Deionized water or distilled water.
4.2.2 Methanol.
5 storage
5.1 The kit is stored at 2~8 °C, do not freeze
5.2 Unused microplates should be sealed and stored dry

6 Precautions

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°C). It is recommended to return to the temperature for at least 2 hours.
6.4 The standard contains aflatoxin B1 (AFB1). Special care should be taken when using it. Gloves should be worn during operation.
6.5 Stop solution contains sulfuric acid, which prevents burns on the skin and corrosive clothing.
6.6 The tips used in different standards and samples should not be mixed, otherwise the test results will be affected.
6.7 The reagents in the different batches of reagent kits must not be mixed; the tips of different standards and samples should not be mixed, otherwise the experimental results will be affected.
6.8 The sample dilution in this kit must be used when diluting the sample, otherwise the results will be affected.
6.9 Avoid foaming when mixing reagents.

7 working fluid preparation

7.1 Aflatoxin B1 (AFB1) standard solution: 0 ppb 0.2 ppb, 0.6 ppb, 1.8 ppb, 5.4 ppb, 16.2 ppb
7.2 Concentrated washing solution: diluted with distilled water at 1:20 (1+19)

7.3 Sample Diluent: Dilute with 1:10 (1+9) in distilled water.
7.3 Color developer: Already reserved, avoid direct light
7.4 Reaction Stop Solution: Already Reserved

8 Sample processing:

(In the extraction process, the sample should be strictly in accordance with the instructions. The extraction process should be accurately diluted. Otherwise, the results will be inaccurate. The sample should be stored in a cool and dark place and stored in cold storage.)

General sample processing

8.1 Take 10g of pulverized sample, add 20ml of 70% methanol solution
8.2 Strong oscillation for 3 minutes
8.3 Filtering with Whatman No 1 filter paper
8.4 Take 100 μl of the treated sample and add 400 μl of sample dilution
8.5 Take 100μl dilution for analysis

Animal tissue pretreatment

8.6 Accurately weigh 1±0.05 g of the homogenized tissue sample into a 50 ml polystyrene centrifuge tube; add 3 ml of acetonitrile-acetone extraction solution, violently shake at 2000 rpm for 20 s and then centrifuge at 4000 r/min for 5 min.

8.7 Take 0.8ml of the supernatant and blow it dry under a nitrogen stream at 50 °C.

8.8 Add 3.2mL sample dilution, vortex at 750rpm for 20s

8.9 take 100ml for analysis

Feed pretreatment method

8.10 Accurately weigh 1±0.05 g of crushed feed sample into 50 ml polystyrene centrifuge tube; add 4ml acetonitrile, 1ml acetone, violently oscillate for 20s at 2000rpm, and centrifuge for 3min at 4000r/min or more.

8.11 Take the supernatant 0.7ml and blow it dry under a nitrogen flow at 50 °C.

8.12 Add 2.8mL sample dilution, vortex for 20s, mix and take 100ml for analysis

Milk pretreatment method

8.13 Take 1 ml of milk sample into 5 ml polystyrene centrifuge tube; add 4 ml sample dilution, mix and take 100ml for analysis

Milk powder pretreatment method

8.14 Accurately weigh 0.3g milk powder sample into 7 ml polystyrene centrifuge tube; add 2ml PBS solution, 2 ml n-hexane, shake and mix

8.154000r/min or more for 5min, remove the organic layer and the intermediate layer, and remove the lower layer solution from 100ml to 400ml.

8.16 after mixing, take 100ml for analysis

9 enzyme-free analysis step

9.1 Instructions for the experiment
9.1.1 Allow all reagents to return to room temperature (25 ± 2 ° C) outside the box for approximately 2 hours before the start of the experiment. After returning to room temperature (25±2°C), remove the microporous strips. Re-seal the excess microporous strips and dry them at 2~8°C. Note: Ensure that the temperature is sufficient, otherwise the accuracy and accuracy of the detection will be affected.
9.1.2 Put the reagent back to 2~8 °C immediately after use.
9.1.3 Please do not change the analysis program
9.1.4 Please use a precise micropipette
9.1.5 Once the operation begins, please do not interrupt any program.
9.1.6 The repeatability of ELISA results depends greatly 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 allow the tip to contact the solution or inner surface of the microwell while loading.
9.2 Analysis steps
9.2.1 Pre-numbering, marking the position of B0, standards and samples, it is recommended to perform two-hole detection
9.2.2 Take the required number of micro-holes (the micro-hole strips are detachable), re-seal the excess slats and immediately return them to 2~8 °C for storage.
9.2.3 Dilute the sample diluent and concentrated washing solution (20×) into working solution (diluted with distilled or deionized water)
9.2.4 Add 50μl of 0.0 ppb standard solution to B0 well
9.2.5 Add 50 μl of standard solution to each standard well.
9.2.6 Add 50μl sample solution to each sample well
9.2.7 Add 50 μl of anti-aflatoxin B1 (AFB1) abzyme conjugate to all wells
9.2.8 Gently shake the plate for a few seconds.
9.3 37 ° C warm bath for 30min (timely tapping the reaction plate during the warm bath can reduce the double hole error)
9.3.1 Remove the liquid from the well and wash the microplate 5 times with the wash solution. The last time should be tapped on the absorbent paper to completely remove the liquid from the well.
9.4 Reaction
9.4.1 Immediately after the washing procedure, add 50 μl of coloring solution A to each microwell with a micropipette, and add 50 μl of coloring solution B; shake the reaction plate slightly to mix thoroughly.
9.4.2 37 ° C warm bath 10min
9.4.3 Add 50 μl of Stop Solution to each well and mix
9.4.4 The absorbance was measured at 450 nm and the result was read within 5 min.

10 result calculation

10.1 Quantitative analysis
10.1.1 The average value (B) of each concentration standard solution and sample absorbance value obtained is divided by the absorbance value (B0) of the first standard (0 standard) and multiplied by 100%, that is, the percent absorbance value.
B—the average absorbance value of the standard solution or sample solution
Average absorbance value of B0-0 ppb standard solution
10.1.2 The logarithm of the concentration of aflatoxin B1 (AFB1) is the X-axis and the percent absorbance is the Y-axis. A standard curve is drawn. According to the percent absorbance of the sample, the abscissa of the corresponding point can be obtained from the curve, which is the logarithm of the concentration of aflatoxin B1 (AFB1), and the antilogarithm is the concentration of aflatoxin B1 (AFB1) in the assay liquid. (ppb)
10.1.3 Since the sample has been pre-diluted, the concentration of the sample 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 compare the sample to the standard absorbance value to determine whether the sample concentration value is less than or greater than the standard value.
10.1.2 Semi-quantitative determination of the instrument: First select an appropriate standard solution to run with the sample, and judge whether the sample concentration value is smaller or larger than the standard value according to the color depth of the sample and the standard.

11 specificity <br>substance cross reaction aflatoxin B1 (AFB1) 100%

12 Kit Parameters <br>The lower limit of detection of this kit is 0.2 ppb
The best value of B0 absorbance should be greater than 1.0
The error in the absorbance of the kit is less than 8%, and the error between the plates is less than 15%.
The recovery rate of the tissue sample extraction method provided by this specification is greater than 80%.
13 standard curve mode (for reference only)
The standard curve provided by the kit ranges from 0.2 ppb to 16.2 ppb.

14 Analytical Limits <br> Samples tested positive for this kit should be confirmed by another method such as HPLC or GC/MS.