So the well is washed to remove anything that is not bound, and the substrate is added to react with the enzyme producing that product that gives off that color. And just like in the case of indirect Eliza, in sandwich Eliza, the darker that color is, the higher the concentration of that antigen. And the lighter the color is, the smaller the concentration of our antigen. Now, if the antigen was not present in our mixture, then this enzyme linked antibody would have nothing to bind to. And so when we wash it in step D, we wash away that enzyme. And so when we add our substrate because the enzyme would not be present in our mixture, that substrate would not be catalyzed, and we would not form that product.