This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Human RANKL antibody. RANKL present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Human RANKL Antibody is added and binds to RANKL in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated RANKL antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Human RANKL. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.