We have developed a two-site ELISA for measurement in serum of bone-related degradation products derived from C-terminal telopeptides of type I collagen. The assay is based on the application of two highly specific monoclonal antibodies against the amino acid sequence of AHD-β-GGR, where the aspartic acid residue (D) is β-isomerized. In a one-step incubation procedure, the degradation products containing cross-linked diisomerized EKAHD-β-GGR peptides are captured by a biotinylated antibody and a peroxidase-conjugated antibody. The generated complex is then bound to the streptavidin surface via the biotin conjugate. Desalted urinary antigens are used for standardization, and parallelism is observed with serum samples. Results are obtained in <2.5 h, and both inter- and intraassay imprecision are <8%. The serum CrossLaps™ concentration was 1748 ± 740 pmol/L (mean ± SD) in premenopausal women (n = 65) and 2952 ± 1325 pmol/L in a group of healthy postmenopausal women (n = 169). The Serum CrossLaps One Step ELISA was capable of detecting a highly significant (P <0.001) effect of hormone replacement therapy in a retrospective study involving 22 postmenopausal women.
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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