In this assay, the test sample initially reacts with the solid phase capture antibody, resulting in the cytokine being bound to the well. The wells are then washed to remove unbound proteins, and biotin-linked antibodies are added to bind to the cytokines. After washing away the unbound antibodies, Streptavidin-HRP conjugate is added to form a complex with the antibody-bound cytokines.

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For colorimetric kits, the HRP substrate, TMB, is added and forms a blue color when the HRP-linked antibodies are detected. The reaction is then terminated with Stop Solution, which changes the color from blue to yellow. The cytokine concentration in each well is directly proportional to its color intensity and can be quantified by measuring its optical density at 450 nm (OD450) in a microplate reader.