To determine the in vitro efficacy of the anti-RANK mAb-sCT conjugate on osteoclast activity, we quantified osteoclast activity using a spectrophotometric total culture TRAP activity assay and a mineral resorption assay. TRAP has been shown to be a specific and sensitive indicator of bone resorption (36) and contributes to the intracellular processing of primary bone matrix degradation products prior to its final release through the basolateral membrane of resorbing osteoclast cells (37, 38) . Osteoclasts, as the bone-resorbing cells of the body, further show the ability to dissolve the mineralized inorganic phase of bone matrix known as hydroxyapatite (39) . We found that the use of our conjugate in culture significantly reduced TRAP enzyme activity as compared to sCT alone, and use of the mAb alone also showed inhibition of TRAP enzyme activity. Although the cause for this effect shown by our anti-RANK antibody alone has not been tested, we speculate that in binding the RANK receptor, the antibody may be functioning as a receptor antagonist. That competitive binding would interfere with the interaction of RANKL with RANK, an essential step of osteoclast differentiation. Nonetheless, further antiresorptive and/or anabolic effector functions could be attributed with the development of a targeted delivery strategy to osteoclast cell receptors. We further evaluated if the mAb-sCT conjugate had an effect on the ability of osteoclasts to resorb bone. We cultured osteoclast-like cells on calcium phosphate-coated culture plates in the presence of conjugate, antibody or sCT. We found that both the conjugate and the mAb alone were capable of significantly reducing the resorption of mineral layer as compared to the sCT alone. Then, we performed cyclic AMP assay to confirm that bioactivity of conjugated calcitonin. Human breast carcinoma T47D cells known to express the calcitonin receptor were used for this purpose. Binding of sCT with its receptor activates adenylyl cyclase, an enzyme responsible for the generation of cAMP. Our in vitro cAMP assay results confirmed that the modification of sCT by conjugation did not interfere with its ability to bind the calcitonin receptor and trigger its biological activity. We measured the conjugate to have increased activity over calcitonin alone, and the exact reason for that effect is still not clear. It is likely our conjugation strategy allowed one mole of antibody to become conjugated with greater than one mole of calcitonin. It has been reported that when IgG is treated with a 10 molar excess of Traut's reagent, 3-7 thiol groups are generated, which may result in the attachment of 3-7 PEGylated calcitonin molecules per molecule of IgG.