The Cortisol (Antigens) in the sample competes with the Antigensic Cortisol conjugated with horseradish peroxidase (HRP) for binding to the limited number of antibodies anti Cortisol coated on the microplate (solid phase) of the Cortisol Saliva ELISA Assay Kit. 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 blu 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. Sample Types: saliva. Range: 0.5 -100 ng/ml. Sensitivity: 0.12 ng/ml. Time: 1.5 hours ? The Eagle Biosciences Salivary Cortisol ELISA Assay Kit is a competitive immunoenzymatic colorimetric method for quantitative determination of Cortisol concentration in saliva.