Specific radioimmunoassay for ovine bone gla-protein (osteocalcin)

Abstract

We developed a sensitive and specific radioimmunoassay for ovine bone gla-protein (osteocalcin) using a polyclonal rabbit antibody raised against ovine bone gla-protein. Bone from lambs was extracted in 0.5 mol/l EDTA and desalted on Sephadex G-25. Bone gla-protein was purified by gel filtration chromatography over Sephadex G-100 and ion-exchange chromatography on DEAE-Sephadex A-25. The protein, subjected to monodimensional electrophoresis migrated as a single spot in SDS PAGE with the same apparent molecular weight of 12 kD as bovine bone gla-protein. The amino acid composition of purified bone gla-protein was in agreement with a previous publication. The competitive RIA uses 125I-labelled bone gla-protein as a tracer and a complex of a second antibody and polyethylene glycol to separate free and antibody-bound 125I-labelled bone gla-protein. The intra- and inter-assay variations are less than 6 and 10%, respectively. There is no reactivity of our antisera with dog sera. The cross-reactivity is only partial with calf and human sera and complete with ovine sera. We measured bone gla-protein levels in serum of 96 normal male sheep of different ages. Serum bone gla-protein rapidly and significantly (P less than 0.001) decreased from 535 +/- 169 microgram/l at birth, to 240 +/- 43 microgram/l at 45 days, 152 +/- 44 micrograms/l at 90 days, and 5.9 +/- 0.7 microgram/l at 7 years of age.(ABSTRACT TRUNCATED AT 250 WORDS)