Human RANKL ELISA Kit (A73880) employs the sandwich enzyme immunoassay technique for the quantitative measurement of human RANKL in serum, plasma, tissue homogenates, and other biological fluids. An antibody specific for RANKL has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the RANKL 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-RANKL Antibody, which binds the captured RANKL 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 RANKL captured in each well. The concentration of RANKL can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.