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The kit assay Rat TNF-α level in the sample, use Purified Rat TNF-α to coat microtiter plate wells, make solid-phase antibody, then add TNF-α to wells, Combine TNF-α antibody with HRP labeled to form antibody-antigen -enzyme-antibody complex, after washing completely, add TMB substrate solution,TMB substrate becomes blue color at HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of TNF-α in the samples is then determined by comparing the O.D. of the samples to the standard curve.