title: A dual-label immunofluorometric assay for human osteocalcin.
abstract: Circulating human osteocalcin (hOC) has been shown to be comprised of two main forms: the intact 1-49 form and the proteolytic N-terminal midfragment (N-mid) spanning amino acid residues 1-43 or 1-44. We used three monoclonal antibodies (MAbs) raised against hOC and bovine osteocalcin in developing a dual-label assay for the simultaneous measurement of the proportions of the intact and N-mid forms in serum samples. The assay is based on time-resolved fluorescence utilizing differently labeled trace MAbs. Biotinylated MAb 2H9 is used as a capture antibody for both the intact hOC and the N-mid. Tracer MAb 6F9 labeled with a Europium (III)-chelate binds to the intact the N-mid and the intact hOC, whereas tracer MAb 3G8 labeled with a Terbium (III)-chelate binds to the intact hOC only. The simultaneous binding of the antibodies was tested by comparing full-length hOC purified from human bone and hOC shortened from the C terminus by four amino acid residues with carboxypeptidase Y. Serum hOC measurements with the dual-label assay were in agreement with the corresponding single-label assays (r = 0.96 for intact + N-mid assay and r = 0.81 for intact assays, n = 91). The lower correlation between the intact assays was attributable to proteolytic susceptibility of the intact form due to one additional freezing and thawing cycle in carrying out the dual-label assay. As measured with the dual-label assay, the levels (mean +/- SD) of serum intact + N-mid OC were 6.2 +/- 2.1 ng/ml in the premenopausal group (n = 44), 13.9 +/- 4.9 ng/ml in the postmenopausal group without hormone replacement therapy (HRT; n = 13), and 7.5 +/- 3.4 ng/ml in the postmenopausal group with HRT (n = 13). The levels of intact hOC in the same groups were 4.8 +/- 1.4 ng/ml, 9.8 +/- 2.9 ng/ml, and 5.3 +/- 2.1 ng/ml, respectively. Whether the main forms of OC or their relative proportions in serum can be used for predicting bone diseases or for monitoring the progression and management of diseases awaits further investigations.
authors.affiliation: Department of Biotechnology, University of Turku, Finland.
authors.forename: A
authors.initials: A
authors.lastname: Ylikoski
authors.forename: J
authors.initials: J
authors.lastname: Hellman
authors.forename: T
authors.initials: T
authors.lastname: Matikainen
authors.forename: S M
authors.initials: SM
authors.lastname: Käkönen
authors.forename: M
authors.initials: M
authors.lastname: Karp
authors.forename: H K
authors.initials: HK
authors.lastname: Väänänen
authors.forename: T
authors.initials: T
authors.lastname: Lövgren
authors.forename: K
authors.initials: K
authors.lastname: Pettersson
mesh_terms: D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D019921:Biotinylation; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry; D005260:Female; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D008593:Menopause / Q000097:blood; D008875:Middle Aged; D015675:Osteocalcin / Q000097:blood* / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013725:Terbium / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D013725:Terbium; D015675:Osteocalcin