This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Fish HIS antibody. HIS present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Fish HIS Antibody is added and binds to HIS in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated HIS 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 Fish HIS. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.