Human Free Prostate Specific Antigen ELISA Kit (A76569) employs the sandwich enzyme immunoassay technique for the quantitative measurement of human Free Prostate Specific Antigen in serum, plasma, tissue homogenates, and other biological fluids. An antibody specific for Free Prostate Specific Antigen has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the Free Prostate Specific Antigen present in each sample is bound to the wells by the immobilized antibody. Following incubation, the wells are washed and then incubated with Biotinylated Anti-Free Prostate Specific Antigen Antibody, which binds the captured Free Prostate Specific Antigen present in each well. Following incubation, unbound biotinylated detection 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 proportional to the amount of Free Prostate Specific Antigen captured in each well. The concentration of Free Prostate Specific Antigen can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.