To quantify the total TRAP activity from in vitro osteoclast cultures, RAW264.7 cells were seeded at a concentration of 2×10 3 cells/well in 96-well culture plates and incubated for 24 h. Peptide factors 50 ng/ml RANKL and MCSF 25 ng/ml were added to the culture, with or without the addition of 100 nM of mAb, 100 nM of mAb-sCT, or 100 nM of sCT. The medium and factors were replaced every 48 h. Osteoclastogenesis was assessed by the spectrophotometric measurement of TRAP activity on day 7. Briefly, the medium was aspirated, and the cell monolayer washed twice with PBS. The cells were then lysed with 100 μl of 0.2% Triton X-100 in water (v/v) for 10 min. TRAP activity in the cell lysate was determined using an Acid Phosphatase Assay Kit (Cayman chemical, USA). The assay utilizes para-nitrophenyl phosphate (pNPP) as a chromogenic substrate for the TRAP enzyme. In the first step, acid phosphatase dephosphorylates pNPP. L-tartrate, an inhibitor of nontartrate resistant acid phosphatase, provided in the kit was used to measure TRAP enzyme activity. In the second step, the phenolic OH-group was deprotonated under alkaline conditions, resulting in p-nitrophenolate that yields an intense yellow color, which was measured at 405 nm using a microplate reader.