Instructions for use of vomitoxin ELISA kit (updated in 2022)

Instructions for vomitoxin ELISA kit

This kit is for research use only

1 Purpose of use:
Qualitative and quantitative analysis of DON in various grains, feed and feed ingredients, corn, wheat and by-products, malt beer and wort, microorganisms, serum, tissue, urine and other samples ( Quantitative detection of vomitoxin) residues
.


2 Experimental principle
This kit adopts the competitive ELISA method.
The microplate is coated with vomitoxin conjugated antigen, the vomitoxin standard or sample is added, and the free vomitoxin and the microwell strip are pre-treated.
The coated vomitoxin conjugated antigens compete with each other for the anti-vomitoxin antibody enzyme label.
The color is developed with TMB substrate.
After adding the stop solution, the color changes from blue to yellow.
The absorbance value is inversely proportional to the content of vomitoxin (vomitoxin) in the sample, and the content of vomitoxin (vomitoxin) in the sample is calculated by the standard curve
.


3 Kit composition
3.
1 Pre-coated vomitoxin (vomitoxin)-conjugated antigen detachable microtiter plate: 1 piece (12 wells × 8 strips)
.

3.
2 Vomitoxin standard: 6 bottles (1ml/bottle), the contents are: 0 ppb, 0.
1 ppb, 0.
3 ppb, 0.
9 ppb, 2.
7 ppb, 8.
1 ppb
.

3.
3 Anti-vomitoxin antibody enzyme conjugate: 1 bottle (6ml)
.

3.
4 Color developer A: 1 bottle (6ml)
.

3.
5 Color developing solution B: 1 bottle (6ml)
.

3.
6 Stop solution: 1 bottle (6ml), 2M sulfuric acid
.

3.
7 Sample Diluent: 1 bottle (10×, 6ml) for sample dilution
.

3.
8 Concentrated washing solution: 1 bottle (20×, 20ml) for plate washing
.

3.
9 A copy of the manual
.

 

4 Materials required but not provided
4.
1 Equipment
4.
1.
1 Wavelength 450nm microplate reader
.

4.
1.
2 Crusher
.

4.
1.
3 Graduated cylinder
.

4.
1.
4 Oscillator
.

4.
1.
5 Funnels
.

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 should be stored at 2~8℃, 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℃), and it is recommended to return to the temperature for at least 2 hours
.

6.
4 The standard product contains vomitoxin (vomitoxin), special attention should be paid when using it, and gloves should be worn during operation
.

6.
5 The stop solution contains sulfuric acid to prevent skin burns and corrosion of clothing during use
.

6.
6 The tips used for different standards and samples cannot be mixed, otherwise the test results will be affected
.

6.
7 Reagents in kits with different batch numbers should not be mixed; tips used in different standards and samples should not be mixed, otherwise the experimental results will be affected
.

6.
8 The sample diluent in this kit must be used when diluting the sample, otherwise it will affect the experimental results
6.
9 Avoid foaming when mixing reagents
.


7 Working solution preparation
7.
1 Vomitoxin standard solution: 0 ppb, 0.
1 ppb, 0.
3 ppb, 0.
9 ppb, 2.
7 ppb, 8.
1 ppb
7.
2 Concentrated washing solution: Dilute with distilled water at 1:20 (1+19) for use
7.
3 Sample diluent: diluted with distilled water at 1:10 (1+10) for use
7.
3 Color developer: ready for use, avoid direct light
7.
4 Reaction stop solution: ready

for use Instructions for operation, the extraction process should be accurately diluted, otherwise the results will be inaccurate, the sample should be stored in a cool, dark place and refrigerated)
8.
1 Take 10g of the crushed sample, add 20ml of 70% methanol solution
8.
2 Strongly shake for 3 minutes
8.
3 Use Whatman No 1 filter paper
8.
4 Take 25µl of the treated sample and add 25µl of sample diluent to the reaction well (the sample dilution factor is 2)

9 Enzyme immunoassay step
9.
1 Experiment instructions
9.
1.
1 Please put all reagents out of the box before the experiment starts Fully return to room temperature (25±2℃), about 2 hours
.

Return the temperature to room temperature (25±2℃) and then take out the microporous strips.
The excess microporous strips are resealed and stored immediately at 2~8℃
.

9.
1.
2 Immediately after use, please put the reagent back to 2~8℃ for storage.

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 depends largely on the operating procedures.
Please operate in strict accordance with the requirements.

9.
1.
7 To avoid cross-contamination, each standard and sample should be loaded with different tips
.
Do not let the tips touch the solution or inner surface of the microwells
9.
2 Analysis steps
9.
2.
1 Pre-number, mark the positions of B0, standard and sample, double well assay is recommended
9.
2.
2 Take the required number of microwells (microwells).
9.
2.
3
Sample Diluent (10×), Concentrated Washing Solution (20×) Dilute into working solution (dilute with distilled water or deionized water) )
9.
2.
4 Add 50µl 0.
0 ppb standard solution to well B0
9.
2.
5 Add 50µl standard solution to each standard well
9.
2.
6 Add 50µl sample solution to each sample well
9.
2.
7 Add 50µl to all wells Anti-vomitoxin antibody enzyme conjugate
9.
2.
8 Gently shake the plate for a few seconds
.

9.
3 Warm bath at 37°C for 30min (dip the reaction plate from time to time during the warm bath to reduce double hole error)
9.
3.
1 Shake off the liquid in the wells, wash the microplate 5 times with washing solution, the last time should be tapped on absorbent paper to completely remove the liquid in the wells
.

9.
4 Reaction
9.
4.
1 Immediately after the washing procedure is completed, add 50µl of chromogenic solution A and 50 µl of chromogenic solution B to each microwell immediately with a micropipette;
9.
4.
2 37℃ Incubate for 10min
.
9.
4.
3 Add 50µl of stop solution to each well and mix well.

9.
4.
4 Measure the absorbance at 450nm, and read the result within 5min
.


10 Calculation of results
10.
1 Quantitative analysis
10.
1.
1 The average value (B) of the absorbance values ​​of each concentration of standard solution and sample obtained in 10.
1.
1 is divided by the absorbance value (B0) of the first standard (0 standard) and multiplied by 100%, namely percent absorbance value
.

B—average absorbance value of standard solution or sample solution B0—average absorbance value of
0ppb standard solution
10.
1.
2 Take the logarithmic value of vomitoxin (vomitoxin) concentration as the X-axis and the percent absorbance value as the Y-axis, draw a standard curve graph
.
According to the percentage absorbance value of the sample, the abscissa of the corresponding point can be obtained from the curve, which is the logarithm of the concentration of vomitoxin, and the logarithm obtained is the concentration of vomitoxin in the test solution C (ppb)
10.
1 .
3 Since 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 sample concentration value is less than or greater than the standard value according to the color depth comparison between the sample and the standard product
.

10.
1.
2 Instrument semi-quantitative determination: First, select an appropriate standard solution to run with the sample, and judge whether the sample concentration value is less than or greater than the standard value according to the comparison of the absorbance values ​​of the sample and the standard product
.

 

11 Specificity
Cross-reaction
of substances Vomitoxin…………………………………………100%

12 Kit parameters
The lower limit of detection of this kit is 0.
05ppb The
best value of B0 absorbance should be greater than 1.
0
kit The error within the absorbance plate is less than 8%, and the error between plates is less than 15%
.

The recovery rate of the tissue sample extraction method provided in this manual is greater than 80%
.


13 The standard curve provided by the kit ranges from 0.
1 ppb to 8.
1 ppb
.


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