Add 100 μl Standard, control or samples to each well.
Incubate 1 hour at RT.

Aspirate and wash 3 times.

Add 100 μl Detection antibody to each well.
Incubate 1 hour at RT.

Aspirate and wash 3 times.

Add 100 μl Substrate solution to each well.
Incubate 10 minutes at RT.

Add 50 μl Stop solution to each well.
Read at 450 nm within 15 minutes.

Calculation