Cortisol is a one-step immunoassay, based on the principle of the competitive method. Cortisol present in the sample and the labeled enzyme-cortisol in the conjugate compete for binding to the capture antibody on the anti-cortisol coated microplate. The enzyme activity in the antibody-bound fraction is inversely proportional to the native cortisol concentration.

The unbound components are removed by washing. After addition of the solution containing TMB and hydrogen peroxide, the wells with bound conjugate develop a blue colour which is converted to yellow after the reaction has been stopped with sulphuric acid.

The colour intensity is inversely proportional to the concentration of the native cortisol in the specimen and can be read at 450 nm.