Measurement of a more stable region of osteocalcin in serum by ELISA with two monoclonal antibodies.
Intact human osteocalcin purified from femoral bones as well as tryptic fragments of the intact molecule [amino acids (aa) 1-19, 20-43, and 45-49] were used to raise and screen monoclonal antibodies (MAbs). A two-site ELISA for measurement of human osteocalcin in serum was developed with use of these MAbs. A MAb recognizing midregion human osteocalcin (aa20-43) was used as capture antibody, and an NH2 terminus (aa7-19)-specific peroxidase-conjugated MAb was used for detection. Human osteocalcin obtained from bone was used for calibration, and parallelism was observed for osteocalcin from serum samples, NH2-terminal midfragments (aa1-43), and synthetic human osteocalcin. Both inter- and intraassay variations were < 7%. Serum osteocalcin in healthy premenopausal women (n = 49) was 18.3 +/- 4.2 micrograms/L (mean +/- SD) and 28.6 +/- 9.7 micrograms/L in early postmenopausal women (n = 114). The mean serum concentration (n = 10) decreased by 10% after 7 days of storage at 4 degrees C, whereas the concentration of intact human osteocalcin was reduced 63%. The N-MID ELISA and an IRMA measuring intact human osteocalcin were used to monitor the effect of hormone replacement therapy in a retrospective study. A significant decrease to the premenopausal concentration was detected only in the N-MID ELISA.
Rosenquist
C
C
Osteometer BioTech A/S, Rødovre, Denmark.
Qvist
P
P
Bjarnason
N
N
Christiansen
C
C
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001842:Bone and Bones / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005260:Female; D006801:Humans; D007202:Indicators and Reagents; D015675:Osteocalcin / Q000097:blood*; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D017698:Postmenopause / Q000097:blood; D017697:Premenopause / Q000097:blood; D012016:Reference Values
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D010446:Peptide Fragments; D015675:Osteocalcin