Free Thyroxine / T4 ELISA Kit (A86983) employs the competitive enzyme immunoassay technique for the quantitative measurement of Free Thyroxine / T4 in serum, plasma, tissue homogenates, and other biological fluids. The 96-well microtiter plate has been pre-coated with Free Thyroxine / T4 antigen. During the incubation, Free Thyroxine / T4 present in the samples or standards competes with the fixed amount of immobilized Free Thyroxine / T4 for binding sites on the Biotinylated Anti-Free Thyroxine / T4 Antibody. The more Free Thyroxine / T4 present in a sample or standard, the less Biotinylated Anti-Free Thyroxine / T4 Antibody that binds to the plate. Following incubation, unbound Biotinylated Anti-Free Thyroxine / T4 Antibody is removed by washing, and an HRP-Streptavidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is inversely proportional to the amount of Free Thyroxine / T4 present in each sample or standard. The concentration of Free Thyroxine / T4 can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.