Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody

In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within- and between-assay CVs. The detection limit was between 0.6 and 1.1 μg/L and the ED₅₀ was 16 μg/L for a 5-μL sample volume. The intra-assay CV over the range 3 to 74 μg/L was ≤ 15%. The interassay CV over the range 3.6 to 46 μg/L was ≤ 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r² = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.