Free Thyroxine ELISA (Free T4 ELISA)

The Free Thyroxine ELISA is a solid phase competitive enzyme immunoassay. Serum samples, standards, and Thyroxine-Enzyme Conjugate Working Reagent are added to wells coated with monoclonal thyroxine antibody. Free thyroxine in the samples and the thyroxine-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 thyroxine 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 HCl, and the absorbance is measured spectrophotometrically at 450 nm. The intensity of the color formed is proportional to the amount of enzyme present and is inversely related to the amount of unlabeled free thyroxine in the sample. By reference to a series of free thyroxine standards assayed in the same way, the concentration of free thyroxine in the unknown sample is quantified.