The synthesized bio-conjugate was initially characterized using SDS-PAGE analysis and ELISA. Protein band above 150 kDa was observed for the conjugate under nonreducing condition of SDS-PAGE analysis, and some bands were seen above 50 kDa and 25 kDa under reducing condition, thus suggesting that conjugation of PEGylated sCT with mAb had occurred. An ELISA further verified the successful conjugation of sCT to our mAb, as the anticalcitonin secondary antibody reagent would only detect immobilized calcitonin residues that were part of the primary anti-RANK conjugates that were binding to the RANK-coated wells of the ELISA plate. A slight signal was observed for PEGylated sCT, and we assume that this could be due to the reaction of thiol reactive MAL functional group of PEGylated sCT with the free thiol group in BSA used as a blocking agent. In the case of conjugate, such MAL groups were utilized in the reaction with free thiol groups generated in antibody.