Add 50 µL of of test specimen to the assigned well, giving a final specimen dilution of 1:2.
Add 50 µL of Non-Reactive Control and Reactive Control I and II to control wells.
Tap gently on all sides of the microplate to ensure proper mixing of the specimens and controls. Carefully cover the microplate with a plate cover to prevent evaporation during incubation.
Incubate for 60±2 minutes at 37±1°C in an incubator (not waterbath for incubation).
Remove and discard the plate cover and wash the microplate with Diluted Plate Wash (1x plate wash).
Blot dry by inverting the microplate and tapping firmly onto absorbent paper. All residual plate wash buffer should be blotted dry.
Add 100 µL of Substrate to wells. Apply a plate cover.
Incubate for 30±2 minutes in the dark at 37±1°C.
Add 50 µL of Stop Solution to each well to stop the color reaction. Tap gently to mix the plate
Read plate at A450/620 nm within 10 minutes.
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