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Clinical Chemistry, Vol 41, 1439-1445, Copyright © 1995 by American Association for Clinical Chemistry
C Rosenquist, P Qvist, N Bjarnason and C Christiansen
Osteometer BioTech A/S, Rodovre, Denmark.
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.
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