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.
Prakash
K V Bhanu
KV
National Institute for Research in Reproductive Health (ICMR), Mumbai 400012, India.
Desai
Meena
M
Bandivdekar
Atmaram H
AH
Donde
Uday M
UM
Khatkhatay
M Ikram
MI
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002417:Cattle; D002457:Cell Extracts / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008970:Molecular Weight; D015675:Osteocalcin / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification*; D011817:Rabbits; D015203:Reproducibility of Results
D000906:Antibodies; D002457:Cell Extracts; D015675:Osteocalcin