Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin

We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 μg/L and the midpoint of the standard curve ranged from 14 to 17 μg/L. The intraassay CV was ≤8% in the range 2.7-52 μg/L; the interassay CV was usually ≤15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently >90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r² = 0.877, slope and intercept (±SE) = 0.88(±0.051) and 0.316(±0.523), respectively]. The range and mean (±SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 μg/L [8.7(±4.4) μg/L] and 1.9-22.8 μg/L [9.1 (±4.4) μg/L], respectively.