The micro ELISA plate provided in the kit has been pre-coated with an specific antibody. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for antigen and Avidin-Horseradish Peroxidase (HRP) conjugate is added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain antigen, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of antigen. You can calculate the concentration of antigen in the samples by comparing the O.D. of the samples to the standard curve.