Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL) is a key cytokine belonging to the TNF superfamily, primarily produced by osteoblasts, activated T cells, and bone marrow stromal cells. It plays a pivotal role in osteoclast differentiation, bone remodeling, and immune regulation by binding to its receptor RANK. RANKL exists in both membrane-bound and soluble forms, with the membrane-bound form exhibiting stronger biological activity. Dysregulation of RANKL is linked to osteoporosis, rheumatoid arthritis, and cancer-induced bone destruction. Therapeutically, RANKL inhibitors like denosumab are used to treat bone loss disorders.To detect the activity of recombinant RANKL, a functional ELISA assay was performed to evaluate the interaction between recombinant mouse RANKL and recombinant human RANK.Briefly, biotin-linked RANK were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to RANKL-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µl stop solution to the wells and read at 450nm immediately. The binding activity of recombinant mouse RANKL and recombinant human RANK was shown in Figure 1, the EC50 for this effect is 0.039 µg/mL.