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This assay employs the quantitative sandwich enzyme immunoassay technique. Amonoclonal antibody specific for TNF-  has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and a TNF- present is boundbythe immobilized antibody. After washing away any unbound substances, anenzyme-linked polyclonal antibody specific for TNF- is added to the wells. Followinga wash to remove any unbound antibody-enzyme reagent, TMB Substrate solution(Chromogenic agent) is added to the wells and color develops in proportion totheamount of TNF- bound in the initial step. The color development is stopped andtheintensity of the color is measured