1. Prepare all reagents, samples, and working standards as instructed.
2. Refer to the Assay Layout Sheet to calculate the number of the wells (including a blank well) to be used and put the remaining wells and the desiccant back into the pouch to seal and store at 4 °C.
3. Add 50 µl of standard or sample (may require dilution) to each well, followed by 50 µl of HRP-conjugated ACE2 (not to the blank well). Shake the plate gently for 1 minute. Seal and incubate for 1 hour at 37 °C.
4. After thorough washing, add 90 μl of TMB Substrate Solution to each well. Incubate for 20 minutes at 37°C.
5. Add 50 µl Stop Solution to each well and gently tap the plate to ensure thorough mixing.
6. Read the OD (optical density) value at 450 nm within 5 minutes using a microplate reader.