Free Estriol ELISA Kit (A74391) employs the competitive enzyme immunoassay technique for the quantitative measurement of universal Free Estriol in serum, plasma, tissue homogenates, and other biological fluids. The 96-well microtiter plate has been pre-coated with Free Estriol antigen. During the incubation, Free Estriol present in the samples or standards competes with the fixed amount of immobilized Free Estriol for binding sites on the Biotinylated Anti-Free Estriol Antibody. The more Free Estriol present in a sample or standard, the less Biotinylated Anti-Free Estriol Antibody that binds to the plate. Following incubation, unbound Biotinylated Anti-Free Estriol 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 Estriol present in each sample or standard. The concentration of Free Estriol can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.