Development of a New Immunoassay for Human Cathepsin K-Generated Periostin Fragments as a Serum Biomarker for Cortical Bone

Patrick Garnero, Nicolas Bonnet, Serge L. Ferrari

Periostin is a matricellular protein mainly expressed by periosteal cells and osteocytes in bone, but is also present in several other tissues. Available immunoassays use antibodies of unclear specificity. The aim of the study was to develop a bone-specific periostin ELISA based on the detection of fragments generated by the osteoclastic and osteocytic protease cathepsin K. In vitro digestion of human recombinant intact periostin by cathepsin K leads to the generation of multiple fragments. Using LS-MS/MS, it was found that the GSLQPIIK peptide was the most efficiently and abundantly generated periostin fragment. A rabbit polyclonal antibody directed against the synthetic GSLQPIIK sequence was produced. Immunohistochemistry experiments of the tibia showed that the GSLQPIIK fragments localized at the periosteal surface and within the osteocytes. Using the same antibody, we developed an ELISA for the measurement of GSLQPIIK in the serum. This ELISA demonstrated intra- and interassay variability below 14% with a sensitivity allowing accurate determinations in the serum of healthy individuals. Serum GSLQPIIK was measured in 160 healthy postmenopausal women (mean age 65 year) participating in the Geneva Retiree Cohort. Serum GSLQPIIK levels did not correlate with total periostin, hip BMD, and the bone markers PINP and CTX. However, GSLQPIIK was negatively correlated (p values ranging from 0.007 to 0.03) with Hr-pQCT measures of tibia and radius cortical bone, but not with trabecular parameters. We have developed the first assay for the detection of periostin fragments generated by cathepsin K. Because serum levels of this new marker significantly correlated with cortical bone measurements in postmenopausal women, it may prove to be useful for the clinical investigation of patients with osteoporosis.