Anti-RANK mAb-sCT conjugate was examined under both reducing and non-reducing conditions of SDS-PAGE analysis, as described above. ELISA was performed, 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. The wells were incubated in triplicate with either mAb alone, functionalized sCT-PEG, or mAb-sCT conjugate for 1 h. The wells were washed with PBST followed by incubation with rabbit anti-salmon calcitonin antibody (US Biologicals) for 1 h. The wells were washed with PBST three times and incubated with HRPO conjugated goat antirabbit IgG (R&D Systems) for 1 h. After final washing with PBST, TMB substrate was added to each well, and the OD measured at 650 nm.