This ELISA kit uses Competitive-ELISA as the method. The microtiter plate provided in this kit has been pre-coated with Cotinine. During the reaction,  Cotinine in the sample or standard competes with a fixed amount of Cotinine on the solid phase supporter for sites on the Biotinylated Detection Ab specific to  Cotinine. Excess conjugate and unbound sample or standard are washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) are added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by adding Stop Solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of  Cotinine in the samples is then determined by comparing the OD of the samples to the standard curve.