All ELISAs were performed in flat-bottomed 96-well plates (Nunc-Immuno MaxisorbTM plates, Nunc). Antibody secretion by hybridoma cells was detected by indirect ELISA. Briefly, 100 μl of antigen (human sRANK receptor) was used for coating at a concentration of 1 μg/100 μl, overnight at 4°C. The wells were washed three times with PBS (pH 7.3) and, to avoid nonspecific binding, incubated with 3% BSA for 1 h at room temperature. After washing, the wells were incubated with 100 μl supernatant from each hybridoma clone for 1 h at room temperature. After washing, bound antibodies were detected using secondary goat anti-mouse IgG conjugated with horseradish peroxidase (GAM-HRPO) at a 1:5000 dilution for 1 h at room temperature. After final washing, 100 μl of 3,3′,5,5′tetramethylbenzidine (TMB substrate) was added to each well and incubated for 15 min at room temperature. The optical density (OD) was measured at 650 nm using an ELISA Vmax kinetic microplate reader (Molecular Devices Corp., California, USA). The clones showing ELISA values five times higher than the negative control were considered positive. Sera of unimmunized mice and irrelevant antibody were used as negative controls. RPMI media and positive sera from hyperimmunized mice were used as blank and positive control, respectively.