Osteoclasts were generated by dosing the RAW264.7 cells with RANKL and MCSF. The staining for TRAP is a technique commonly used to visualize osteoclasts. The principle behind staining of TRAP involves the use of napthol AS phosphates in conjunction with fast garnet GBC salts for the detection of acid phosphatase. This diazonium fast garnet GBC salt was selected because it couples rapidly at acid pH, forming insoluble dye deposits. Napthol AS-BI, released by enzymatic hydrolysis, couples immediately with fast garnet GBC salt, resulting in the Fig. 3 (a) ELISA conducted on RANK receptor-coated plate. The optical density was seen to be proportional with the concentration of anti-RANK antibody. (b) ELISA of the mAb-sCT conjugate where anti-Calcitonin secondary antibody reagent was used to detect immobilized Calcitonin residues that were part of the primary anti-RANK conjugates and that were binding to the RANK-coated wells. Optical density for mAb alone was negligible as it did not have calcitonin to be detected by anti-calcitonin antibody. Similarly, OD for sCT-PEG was also significantly low as it did not have anti-RANK antibody to bind to RANK-coated plate.