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 ELISA 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.
3. Add 100 μL of the sample diluted solution to the blank wells; add 100 μL of the negative and positive
reference solution to the negative and positive wells respectively; add 100 μL of the diluted sample to each
of the sample well.
4. Mix well, cover the membrane, and place at 37ºC (recommended water bath) in the dark for 30 minutes.
5. Shake off the liquid in the well, add 350μ L of working washing liquid to each well, let it stand for 30
seconds and discard it, repeat washing 5 times, and finally pat dry with clean absorbent paper.
6. Add 100 μL of enzyme marker to each well (except blank wells). Cover the cover film, set at 37°C, and
avoid light for 30 minutes.
7. Wash, same as step 5.
8. Add 50 μL of substrate solution and coloring solution to each well in sequence, mix, cover the cover plate
film, set at 37°C, and avoid light for 10 minutes.
9. Add 50μL of stop solution to each well, mix, and measure the absorbance (OD value) of each well at
450nm (630nm can be used as the reference wavelength). If there is no reference wavelength, use a blank
well to adjusting zero (The absorbance of all wells minus the absorbance of blank).
1. The sample OD value ≥ 0.38 is positive; the sample OD value between 0.2 and 0.38 is suspicious; the
sample OD value <0.2 is negative.
2. When the result is negative or suspicious, it indicates that the antibody level of the pig is insufficient, and
it is recommended to vaccinate accordingly.
3. Under normal conditions, the negative ≤0.10 and the positive ≥0.6.