Operating Procedures:

1.      Temperature Balancing: Take the testing kit from the refrigerator then wait for 30 minutes for it to reach the room temperature.

2.      Solution Perpetration: Carry out 20x dilution of the concentrated washing solution using distilled or deionized water and mix well it well prior to use.

3.      Adding Sample: Leave an empty well without adding any liquid (leave 3 wells for negative control and 2 wells for positive control and 2 wells for cutoff control). Add 100μl each of above mentioned controls to their corresponding wells. Add 100μl of sample diluent to the rest of the wells prior to orderly adding 10μl of testing sample to each well and mix well.

4.      Incubation: Apply the cover membrane to cover the microplate, and put it into a 37℃ water bath or an incubator for 30 minutes.

5.      Washing: Discard all the solution from the microplate fill up each will with washing solution. Wait for 30 seconds and discard all the solution from the microplate. Repeat the washing step 5 times and pat dry the microplate with a paper towel. Alternatively, use a micro plate washer to wash the plate 5 times and pat dry the plate with a paper towel.

6.      Add Enzyme: Add 100ml enzyme complexes to each well (apart from the controls).

7.      Incubation: Cover the microplate with the cover membrane and incubate at 37℃±1℃ for 30 minutes.

8.      Wash Microplate: Repeat step 6.

9.      Coloration: Add 50ml each of substrate A and B to each well (including the empty control wells), cover the microplate with a cover membrane and mix well by oscillating. Incubate at 37℃±1℃ in the dark for 15 minutes.

10.  Stop: Add 50μl of stop solution to each well mix well by oscillating.