Competitive immunoenzymatic colorimetric method for quantitative determination of free Cortisol concentration in Urine. This product is for Research Use Only; International customers contact your representative to receive information about the CE marked version. The Cortisol (antigen) in the sample competes with the antigenic Cortisol conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti Cortisol coated on the microplate (solid phase). After incubation, the bound/free separation is performed by a simple solid-phase washing. Then, the enzyme HRP in the bound-fraction reacts with the Substrate (H2O2) and the TMB Substrate and develops a blue color that changes into yellow when the Stop Solution (H2SO4) is added. The colour intensity is inversely proportional to the Cortisol concentration of in the sample. Cortisol concentration in the sample is calculated through a calibration curve.