Principle of Assay
Human TNF alpha ELISpot Kit (A341836) is a sandwich ELISA designed for the sensitive and quantitative measurement of human TNF alpha in peripheral blood mononuclear cells (pbmcs). The assay utilizes a 96-well microtiter plate pre-coated with an antibody specific for TNF alpha. Standards and samples are added to the wells, allowing TNF alpha present in the samples to bind to the immobilized capture antibody. After washing, a biotinylated detection antibody specific for TNF alpha is added, forming an antibody–antigen–antibody complex. Unbound detection antibody is removed by washing, followed by the addition of streptavidin conjugated to horseradish peroxidase (HRP). Following incubation and washing, TMB substrate is added, resulting in a colorimetric reaction that produces a blue color which turns yellow upon addition of stop solution. The intensity of the color is directly proportional to the concentration of TNF alpha in the sample. The concentration of TNF alpha is determined by measuring the absorbance at 450 nm and comparing the results to the standard curve.
