1. Analyte capture
Provided microplate is precoated with capture antibody(Ab). Any analyte ( red dots)in the standard or samples added to the well will be captured. Unbound molecules are washed away.
2. HRP-detection Ab binding to captured analyte
A second antibody conjugated with HRP (blue shield) is added and binds to the captured Analyte. Free detection antibody Is washed away.
3. HRP/substrate color display
HRP substrate TMB (Tetramethylbenzidine) is added to the wells. Blue color is developed proportionally to the amount of analyte in the sample. Blue color development is stopped and turns to yellow when the Stop Solution is added. OD450 is measured to make standard curve and calculate analyte concentration.