The ELISA is based on the the qualitative enzyme immunoassay technique. The microplate provided in this kit has been pre-coated with an antigen specific to Brucella IgM, make it to solid-phase antigen.Samples are added to the microplate wells and combined to the specific antigen. Then a Horseradish Peroxidase (HRP)-conjugated antigen specific for Brucella IgM is added to each Microplate well and incubated,so the antigen-antibody-Enzyme labeled antigen complex is formed.Following a wash to remove any unbound reagent, then theTMB substrate solution is added to each well. Only those wells that contain Brucella IgM and HRP conjugated Brucella antigen will appear blue in color and then turn yellow after the addition of the stop solution. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm. The qualitative determination of Brucella IgMis determinedbycomparing with the CUTOFF value.