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

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 osteo...

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Published in:Clinical chemistry (Baltimore, Md.) Md.), 2000-11, Vol.46 (11), p.1751-1754
Main Authors: Alatalo, Sari L, Halleen, Jussi M, Hentunen, Teuvo A, Monkkonen, Jukka, Vaananen, H. Kalervo
Format: Article
Language:English
Subjects:
Acid Phosphatase - immunology
Acid Phosphatase - metabolism
Animals
Biological and medical sciences
Bone Marrow Cells - cytology
Bone Marrow Cells - enzymology
Bone Marrow Cells - metabolism
Bones, joints and connective tissue. Antiinflammatory agents
Cell Differentiation
Cells, Cultured
Humans
Immune Sera
Immunoassay
Isoenzymes - immunology
Isoenzymes - metabolism
Medical sciences
Mice
Osteoclasts - cytology
Osteoclasts - enzymology
Osteoclasts - metabolism
Pharmacology. Drug treatments
Rabbits
Tartrate-Resistant Acid Phosphatase
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Summary: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
ISSN:0009-9147
1530-8561
DOI:10.1093/clinchem/46.11.1751