Extraction, Purification, and Development of an Enzyme‐Linked Immunosorbent Assay for Osteocalcin

Abstract

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 5.8 kDa, 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 µg 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.