Rapid Screening Method for Osteoclast Differentiation in Vitro That Measures Tartrate-resistant Acid Phosphatase 5b Activity Secreted into the Culture Medium

Abstract

Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) 5b into the circulation. We studied the release of TRAP 5b from osteoclasts using a mouse in vitro osteoclast differentiation assay. We developed and characterized a polyclonal antiserum in rabbits, using purified human osteoclastic TRAP 5b as antigen. The antiserum was specific for TRAP in Western analysis of mouse osteoclast culture medium and was used to develop an immunoassay. We cultured mouse bone marrow-derived osteoclast precursor cells for 3-7 days with or without clodronate in the presence of vitamin D and analyzed the number of osteoclasts formed and the amount of TRAP 5b activity released into the culture medium. TRAP 5b activity was not secreted from osteoclast precursor cells. Addition of clodronate-containing liposomes decreased in a dose-dependent manner the number of osteoclasts and TRAP 5b activity released in 6-day cultures. The amount of TRAP 5b activity in the medium detected by the immunoassay correlated significantly with the number of osteoclasts formed (r = 0.94; P<0.0001; n = 120). The TRAP 5b immunoassay can be used to replace the laborious and time-consuming microscopic counting of osteoclasts in the osteoclast differentiation assay and to test the effects of potential therapeutic agents on osteoclast differentiation, enabling fast screening of large amounts of potential therapeutic agents.