This ELISA kit uses Competitive-ELISA as the method. The microtiter plate provided in this kit has been pre-coated with HIS. During the reaction,  HIS in the sample or standard competes with a fixed amount of HIS on the solid phase supporter for sites on the Biotinylated Detection Ab specific to  HIS. 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  HIS in the samples is then determined by comparing the OD of the samples to the standard curve.