1. Leave the kit at room temperature for more than 30 minutes before use and return to room temperature
(20-25°C).
2. Take the required amount of enzyme-labeled slats, set up 1 well for the blank and 2 wells for the
negative/positive separately, seal the unused slats as soon as possible and store at 2~8ºC. Wash twice with
diluted washing solution.
3. Add 50 μL of the sample diluted solution to the blank wells; add 50 μL of the negative and positive
reference solution to the negative and positive wells respectively; add 50 μL of the enzyme marker to each
wwell, mix gently by shaking, and incubate at 37°C for 30 minutes.
4. Discard the reaction solution, add 200 μL of washing solution to each well, and do not need to stand.
Shake off the washing solution from the well, repeat washing 5 times, and finally pat dry on clean absorbent
paper.
5. Add 100 μL of substrate solution to each well and coloring for 15 minutes at room temperature in the
dark.
7. Add 50 μL of stop solution to each well and mix well. Within 10 minutes, use 450 nm wavelength (630
nm can be used as reference wavelength) to measure the absorbance (OD value) of each well. If there is no
reference wavelength, use a blank well to adjusting zero (The absorbance of all wells minus the absorbance
of blank)
Results:
The test condition is that the difference between the average OD value of the negative reference well and the
positive reference is ≥0.5.
formula: Y=Sample well OD/ the average OD of negative well
When the result is negative or suspicious, it indicates that the pig antibody level is insufficient, and
recommend to vaccinate accordingly