The fT3 test is a solid phase competitive enzyme immunoassay. Patient serum samples, standards, and T3-Enzyme Conjugate Working Reagent are added to wells coated with monoclonal T3 antibody. fT3 in the patient specimen and the T3 labeled conjugate compete for available binding sites on the antibody. After a 60 minute incubation at room temperature, the wells are washed with water to remove unbound T3 conjugate. A solution of H2O2/TMB is then added and incubated for 20 minutes, resulting in the development of blue color. The color development is stopped with the addition of 3N HCI, 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 fT3 in the sample. By reference to a series of fT3 standards assayed in the same way, the concentration of fT3 in the unknown sample is quantified.