ELISA was performed as above, using 100 μl RANK receptor for coating at a concentration of 1 μg/100 μl, overnight at 4°C. To avoid nonspecific binding, the wells were incubated with 3% BSA for 1 h at room temperature. After washing, the wells were incubated with different concentrations of antibody for 1 h. The wells were washed with PBS, and the bound antibodies were detected using secondary antibody (goat anti-mouse IgG conjugated with horseradish peroxidase) for 1 h. After final washing, 100 μl of TMB substrate was added to each well and incubated for 15 min. The optical density (OD) was measured at 650 nm.