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 enzy me-linked immunosorbent assay (ELISA) on microtiter plates for osteoca lcin in human serum with use of an antibody raised against human osteo calcin. We used a monoclonal antibody against bovine osteocalcin as th e capture antibody; the second antibody was a polyclonal antibody agai nst human osteocalcin. The amount of bound second antibody was determi ned with use of swine anti-rabbit antibody labeled with horseradish pe roxidase. We demonstrated independence of volume and determined the re covery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 mug/L and the midpoint of the standard curve ranged from 14 to 17 mug/L The intraassay CV was less-than-or-equal-to 8% in the range 2.7-52 mug/L; the interassay CV was usually less-than-or-equal-to 15% in the same ra nge. Analytical recovery of human osteocalcin standard added to serum samples was consistently > 90%. Values for osteocalcin measured in ser um from 44 normal subjects were similar to those obtained with a compe titive enzyme immunoassay (EIA) that used a monoclonal antibody agains t bovine osteocalcin. There was a good correlation between the two ass ays [r2 = 0.877, slope and intercept (+/-SE) = 0.88(+/-0.051) and 0.31 6(+/-0.523), respectively]. The range and mean (+/-SD) for the sandwic h ELISA and the competitive EIA were 1.7-18.1 mug/L [8.7(+/-4.4) mug/L ] and 1.9-22.8 mug/L [9.1(+/-4.4) mug/L], respectively.