Principle of Assay
Human Perforin ELISpot Kit (A341835) is a sandwich ELISA designed for the sensitive and quantitative measurement of human Perforin in peripheral blood mononuclear cells (pbmcs). The assay utilizes a 96-well microtiter plate pre-coated with an antibody specific for Perforin. Standards and samples are added to the wells, allowing Perforin present in the samples to bind to the immobilized capture antibody. After washing, a biotinylated detection antibody specific for Perforin 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 Perforin in the sample. The concentration of Perforin is determined by measuring the absorbance at 450 nm and comparing the results to the standard curve.
