This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Human Cotinine antibody. Cotinine present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Human Cotinine Antibody is added and binds to Cotinine in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated Cotinine antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Human Cotinine. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.