Principle of Assay
Human CD25 ELISA Kit (A117469) employs the sandwich enzyme immunoassay technique for the quantitative measurement of human CD25 in serum, plasma and cell culture supernates. An antibody specific for CD25 has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the CD25 present in each sample is bound to the wells by the immobilized antibody. Following incubation, the wells are washed and then incubated with Biotinylated Anti-CD25 Antibody, which binds the captured CD25 present in each well. Following incubation, unbound biotinylated detection antibody is removed by washing, and an HRP-Streptavidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, TMB substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is proportional to the amount of CD25 captured in each well. The concentration of CD25 can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.
