1. The kit is placed at room temperature for 30 minutes. The 20-fold concentrated lotion is diluted 20-fold with
deionized or distilled water.
2. Add 100μL of sample diluent and 5μL of sample to each well of the test wells, mix gently; one well of blank
reference without liquid; two wells of positive and negative reference, add positive reference serum and negative
reference serum respectively 100μL/well. After attaching the sealing film, place the box in a 37°C water bath or a
constant temperature incubator and incubate for 30 minutes.
3. Discard the sealing film and wash the plate with a plate washing machine or hand, so that each wells is filled with
the washing liquid without overflowing (about 300 μL), the soaking time is 10 seconds, the plate is washed 5 times,
and finally in absorbent paper dry up.
4. Add 100μL of enzyme working reference solution to each well (except the blank wells), After attaching the sealing
film, place the box in a 37°C water bath or a constant temperature incubator and incubate for 30 minutes.
5. As in the above procedure 3, wash the plate and dry.
6. Add 50μL of substrate solution and coloring solution to each well in sequence, gently shake and mix well. After
attaching the sealing film, place the box in a 37°C water bath or a constant temperature incubator, and incubate for 10
minutes in the dark.
7. Discard the sealing film, add 50μL of stop solution to each well, gently shake to mix, measure the OD value with a
microplate reader at a wavelength of 450nm (adjust to zero with blank wells), and print the results.
Results:
1. Sample OD value ≥ Cut Off value, judged as positive, sample OD value < Cut Off value, judged as negative.2. Cut Off=3.1×Average OD value of negative reference (when the average OD value of negative is <0.05, it is
calculated as 0.05; when the average OD value of negative is ≥0.05, it is calculated as the actual value).
3. Under normal circumstances, the negative <0.10, the positive ≥ 1.0.