Radioimmunoassay for human osteocalcin using an antibody raised against the synthetic human (h37-49) sequence

Radioiodination of synthetic human 37-49 osteocalcin requires optimal labeling conditions in order to obtain a maximum of mono- and di-iodinated tracer with little contamination by tri- and tetra-iodinated products or "radio-damage." The antibody raised against osteocalcin(h37-49) had the highest affinity for the C-terminal peptide used for iodination and the larger peptide (h30-49). The intact bovine osteocalcin (b1-49) revealed less immunoreactivity. This C-terminal specific radioimmunoassay detected the intact human osteocalcin in HPLC purified plasma and peritoneal dialysate from patients with terminal renal insufficiency and in extracted human bone. Some quantities of osteocalcin Peptides with a higher hydrophobicity were predominantly detected in uremic plasma. These Peptides that had a higher molecular weight than the intact human molecule might represent aggregated forms of the intact bone-derived osteocalcin. Immunoreactivity in plasma samples from healthy individuals revealed a remarkable difference as to which substance was employed for anticoagulation. Compared to heparin, the addition of EDTA largely reduced the osteocalcin immunoreactivity, implying that conformational changes within the N-terminal portion (containing the Gla- and Cys-residues) are extended to the C-terminal portion.