Rapid screening method for osteoclast differentiation in vitro that measures tartrate-resistant acid phosphatase 5b activity secreted into the culture medium
Sl. Alatalo et al., Rapid screening method for osteoclast differentiation in vitro that measures tartrate-resistant acid phosphatase 5b activity secreted into the culture medium, CLIN CHEM, 46(11), 2000, pp. 1751-1754
Background: 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.
Methods: We developed and characterized a polyclonal antiserum in rabbits,
using purified human osteoclastic TRAP 5b as antigen. The antiserum was spe
cific for TRAP in Western analysis of mouse osteoclast culture medium and w
as used to develop an immunoassay. We cultured mouse bone marrow-derived os
teoclast precursor cells for 3-7 days with or without clodronate in the pre
sence of vitamin D and analyzed the number of osteoclasts formed and the am
ount of TRAP 5b activity released into the culture medium.
Results: 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 cul
tures. The amount of TRAP 5b activity in the medium detected by the immunoa
ssay correlated significantly with the number of osteoclasts formed (r = 0.
94; P < 0.0001; n = 120).
Conclusions: The TRAP 5b immunoassay can be used to replace the laborious a
nd time-consuming microscopic counting of osteoclasts in the osteoclast dif
ferentiation assay and to test the effects of potential therapeutic agents
on osteoclast differentiation, enabling fast screening of large amounts of
potential therapeutic agents. (C) 2000 American Association for Clinical Ch
emistry.