Extraction, purification, and development of an enzyme-linked immunosorbent assay for osteocalcin

The present study describes the isolation and purification of osteocalcin (OC) from bovine bones and the development of an enzyme-linked immunosorbent assay (ELISA) for OC as a marker of bone formation, for assessing bone health. Bone proteins were extracted from about 90 g of bovine bone powder using 20% formic acid. The protein extract was fractionated by gel permeation chromatography on Sephadex G-50 column followed by fast protein liquid chromatography (FPLC) on a MONO-Q column. The immunoreactive active fraction was then purified by chromatofocusing, using FPLC on a MONO P column and a single homogeneous band of molecular size of about 5.8kDa, as judged by Tricine SDS-PAGE following silver staining of the gel, was obtained. It reacted specifically with its antibodies in an ELISA. About 678 microg of purified OC was yielded from about 90 g of bovine bones. The purified OC was subsequently used for the raising antisera, which was used in the development of an indirect ELISA. The developed ELISA has a sensitivity of 2.5-4.0 ng/mL and was used in estimating levels of OC in women of various age groups.