In the T4 EIA, a certain amount of anti-T4 antibody is coated on microtiter wells. A measured amount of patient serum, and a constant amount of T4 conjugated with horseradish peroxidase are added to the microtiter wells. During incubation, T4 and conjugated T4 compete for the limited binding sites on the anti-T4 antibody. After a 60 minutes incubation at room temperature, the wells are washed 5 times by water to remove unbound T4 conjugate. A solution of TMB Reagent is then added and incubated for 20 minutes, resulting in the development of blue color. The color development is stopped with the addition of Stop Solution, and the absorbance is measured spectrophotometrically at 450nm. The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled T4 in the sample. By reference to a series of T4 standards assayed in the same way, the concentration of T4 in the unknown sample is quantified
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