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 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was < or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually < or = 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 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.
Department of Biochemistry, University College Galway, Ireland.
D A
DA
Monaghan
M J
MJ
Power
P F
PF
Fottrell
D000818:Animals; D000906:Antibodies; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D005260:Female; D006801:Humans; D015675:Osteocalcin / Q000097:blood* / Q000276:immunology; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology; D011786:Quality Control; D012016:Reference Values
D000906:Antibodies; D010446:Peptide Fragments; D015675:Osteocalcin; D002118:Calcium