Receptor activator of nuclear factor kappa-B ligand (RANKL), also known as tumor necrosis factor ligand superfamily member 11 (TNFSF11), TNF-related activation-induced cytokine (TRANCE), osteoprotegerin ligand (OPGL), and osteoclast differentiation factor (ODF), is a protein that in humans is encoded by the TNFSF11 gene. RANKL is a member of the tumor necrosis factor (TNF) cytokine family, it binds to RANK on cells of the myeloid lineage and functions as a key factor for osteoclast differentiation and activation. It may also bind to osteoprotegerin, a protein secreted mainly by cells of the osteoblast lineage which is a potent inhibitor of osteoclast formation by preventing binding of RANKL to RANK. Cluster Of Differentiation 40 Ligand (CD40L) has been identified as an interactor of RANKL, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant rat RANKL and recombinant human CD40L. Briefly, RANKL were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 ul were then transferred to CD40L-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-RANKL pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 5 times. With the addition of substrate solution , wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450 nm immediately. The binding activity of recombinant rat RANKL and recombinant human CD40L was shown in Figure 1, the EC50 for this effect is 0.015 ug/mL.