Measurement of a more stable region of osteocalcin in serum by ELISA with two monoclonal antibodies

Abstract

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.

Effects of Simvastatin on Bone Turnover and BMD: A 1-Year Randomized Controlled Trial in Postmenopausal Osteopenic Women

“…Antibodies that recognize both the intact (1-49) and the amino-terminal fragment (N-Mid-osteocalcin; 1-43) were used. (19) For both osteocalcin and CTx, the total CV was Ͻ6%. Alkaline phosphatase (ALP) and bone-specific ALP (bone-ALP) were measured spectrophotometrically using an automated instrument (Hi-…”

Dose‐effect relations of loop‐ and thiazide‐diuretics on calcium homeostasis: a randomized, double‐blinded Latin‐square multiple cross‐over study in postmenopausal osteopenic women

“…The inter-and intra-assay CV were 10% and 8%, respectively. Plasma osteocalcin was measured by ELISA using an automated instrument (Elecsys, 2010 immunoassay analyzer, Roche Diagnostics, Basel, Switzerland) with antibodies that recognise both intact-(1-49) and N-Mid-osteocalcins (1-43) [23]. The total CV was less than 5%.…”

Association of Simvastatin and Hyperlipidemia With Periodontal Status and Bone Metabolism Markers

“…27,28 Serum osteoprotegerin (OPG) and osteocalcin (OCN) concentrations were quantified with an enzyme-linked immunosorbent assay. ** 29,30 Samples were analyzed by the Clinical Analysis Department of the San Cecilio University Hospital, Granada, Spain, which provided the reference values.…”