In this assay, cytokines present in the sample bind to specific capture antibodies pre-coated on a 96-well plate. After unbound components are washed away, biotin-labeled detection antibodies are added to bind the captured cytokines. Streptavidin-HRP is then introduced, forming a complex with the biotin-labeled antibodies.

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• Colorimetric kits: TMB substrate is added, producing a blue color in the presence of HRP. The reaction is stopped with Stop Solution, turning the color yellow. The intensity of the color is measured at an absorbance of 450 nm and is directly proportional to the amount of cytokine present.

• Chemiluminescent kits: A luminescent HRP substrate is added, and the resulting signal is measured as relative light units (RLUs) using a plate luminometer. The luminescent intensity reflects the concentration of each cytokine.

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This method enables simultaneous and quantitative detection of multiple cytokines in a single experiment.