A DUAL-LABEL IMMUNOFLUOROMETRIC ASSAY FOR HUMAN OSTEOCALCIN

Circulating human osteocalcin (hOC) has been shown to be comprised of two main forms: the intact 1-49 form and the proteolytic N-terminal mi dfragment (N-mid) spanning amino acid residues 1-43 or 1-44, We used t hree monoclonal antibodies (MAbs) raised against hOC and bovine osteoc alcin in developing a dual-label assay for the simultaneous measuremen t of the proportions of the intact and N-mid forms in serum samples, T he assay is based on time-resolved fluorescence utilizing differently labeled tracer MAbs, Biotinylated MAb 2H9 is used as a capture antibod y for both the intact hOC and the N-mid, Tracer MAb 6F9 labeled with a Europium(III)-chelate binds to 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 co mparing full-length hOC purified from human bone and hOC shortened fro m the C terminus by four amino acid residues with carboxypeptidase Y. Serum hOC measurements with the dual-label assay were in agreement wit h the corresponding single-label assays (r = 0.96 for intact + N-mid a ssays and r = 0.81 for intact assays, n = 91), The lower correlation b etween the intact assays was attributable to proteolytic susceptibilit y 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 th e main forms of OC or their relative proportions in serum can be used for predicting bone diseases or for monitoring the progression and man agement of diseases awaits further investigations.