Weigh
Weigh 5g samples through 20 mesh screens
Add extraction solution
Add 25ml of 70% methanol
Vortex oscillation
Vortex oscillation for 3 minu
Centrifugal
Centrifuge at 4000 RPM for 5 min
Take the supernatant
Take 100 μl of sample supernatant
Sampling
Add 50 μl of sample solution, ELISA reagent, and antibody working solution separately
Wash plate
Wash the ELISA plate 4 times
Add substrate
Add 100 μl of substrate/chromogen
Add stop solution
Add 100 μl of stop solution
Reading
Values are read using an enzyme marker
T-2 toxin ELISA Quantitative Test Kit Operation guidance
1. Sample pre-processing
1. Take 2kg of representative samples for grinding, so that more than 95% of the samples can pass
through a 20 mesh
sieve. Mix evenly for sampling.
2. Preparation of extraction solution (70% methanol): According to the dosage, mix methanol and
distilled water in a
7:3 volume ratio.
3. Weigh 10g ± 0.1g of the above sample into a clean container with a lid, and add 50mL of 70%
methanol (the ratio of
sample to 70% methanol is 1:5 (w: v)).
4. Fully shake and extract for 3 minutes, let it stand and take the supernatant. Centrifuge at 4000
rpm for 5
minutes.
5. Take 100 μl of supernatant and add 900 μl of T-2 toxin sample diluent (10 fold dilution), mix well and
wait for testing.
2. Sample testing
1. Premixing: take appropriate amount of premixed Flat noodles as required, add 60 μ L of T-2 toxin
calibrator/sample to
be tested into the micropore and record its position. Add 120 μl of T-2 toxin enzyme-linked immunosorbent
assay (ELISA) reagent to each well, pipette and mix well or shake horizontally.
(A ratio of 1:2 is sufficient for the pre mixing of calibration sample/test sample and enzyme reagent.
To make a re
well, mix 100 μl of calibration sample/test sample with 200 μl of enzyme, and add 100 μl to
each of the two detection plate wells.)
2. Sample addition: Add 100 μl of pre mixed liquid to the detection plate hole. Cover with cover
film and
incubate at 25 ℃ for 15 minutes.
3. Washing plate: shake off the solution in the micropores, fill with distilled water, repeat 4
times, and pat dry
on absorbent paper.
4. Color development: Add 100 μl of color development solution to each well, cover with a cover
film, and
incubate at 25 ℃ in the dark for 5 minutes.
5. Termination: Add 100 μl of termination solution to each well, and immediately read the absorbance
value
at 450/630nm dual wavelength.
3.Experimental operation tips
Doing replicate holes for the calibration product and the measured sample can improve the accuracy of
thedetection.
Replace the pipette tip when drawing different reagents to prevent cross-contamination.
When using the pipette, be careful to avoid drawing in bubbles and affecting the volume of the added
liquid. The way of drawing once and dispensing twice is easy to splatter after dispensing the liquid,
so it is recommended to use the way of drawing twice and dispensing once to add the liquid vertically
to the center of the micropores.
The liquid should be dispensed quickly and should not form water droplets.
The transfer of the mixed liquid in the pre-mixing plate to the detection plate starts the reaction.
To reduce the error, please control the time difference between the transfer of different micropores
(increase the transfer speed,control the number of single-sample tests. It is better to use a
multi-channel pipette to test 4 strips at a time;it is better to use a single-channel pipette to test
2 strips at a time).
Do not use expired kits, and do not mix different batches of reagents.
Please keep the kit properly. Freezing, room temperature, and high temperature conditions will
accelerate the aging of the kit. In use, please avoid cross-contamination of different reagents. If an
abnormality of the reagent is found, such as the chromogenic solution turning blue, or the reagent
being turbid, do not use it.
This method is only for the rapid preliminary screening of samples. For confirmation, please refer
to methods such as liquid chromatography.
