Osteoprotegerin (OPG), a secreted glycoprotein belonging to the tumor necrosis factor (TNF) receptor superfamily, is a crucial decoy receptor involved in the regulation of bone metabolism. It is primarily produced by osteoblasts and functions as a key inhibitor of osteoclastogenesis—the formation of bone-resorbing osteoclasts. By binding to its natural ligand, RANKL, OPG prevents RANKL from interacting with its functional receptor, RANK, on osteoclast precursors. This inhibition effectively blocks the signaling pathway essential for osteoclast differentiation and activation, thereby protecting bone from excessive resorption and maintaining skeletal integrity. OPG's role extends beyond bone, implicating it in vascular and immune biology.To detect the activity of recombinant OPG , a functional ELISA assay was performed to evaluate the interaction between recombinant human OPG and recombinant rat RANkL.Briefly, OPG was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to RANkL-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-OPG pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, 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 450/630nm immediately. The binding activity of recombinant human OPG and recombinant rat RANkL was shown in Figure 1, the EC50?for this effect is 0.24μg/mL..