DEVELOPMENT OF A HETEROLOGOUS RADIOIMMUNOASSAY FOR CANINE OSTEOCALCIN

The aim of this study was to develop a routine and reliable radioimmun oassay (RIA) for dog osteocalcin. Two peaks of dog osteocalcin were pu rified to apparent homogeneity according to N-terminal sequence analys is. Amino acid composition analysis suggested that the second peak was intact dog osteocalcin whereas the first peak could be a truncated mo lecule. High titer (>1:5,000) anti-dog osteocalcin antisera were produ ced in rabbits. The antiserum recognized dog and rat osteocalcins but not that in serum of human, bovine, rabbit, mouse, guinea pig, or goat . A homologous RIA using anti-dog osteocalcin as the antibody and dog osteocalcin as the tracer and standard was developed. Taking advantage s of the facts that (1) anti-dog osteocalcin crossreacted in parallel with rat osteocalcin and (2) purified rat osteocalcin is commercially available, we devised an approach that used rat osteocalcin as the tra cer and standard, and anti-dog osteocalcin as the antibody to develop a heterologous RIA. This assay recognized dog serum osteocalcin and di luted in parallel with rat and dog osteocalcins. Quantitation was done using rat osteocalcin to construct standard curves, and results were expressed in ng/ml of rat osteocalcin-equivalent. The detection limit of the assay was 5 ng/ml rat osteocalcin-equivalent, and half-maximal displacement was seen at 30-40 ng/ml rat osteocalcin-equivalent. The i nter- and intraassay variations were 16.1% and 8.5%, respectively. The assay accurately determined the amount of exogenously added dog osteo calcin in serum. The results quantitated with this RIA correlated well (r = 0.975, n = 86) with those obtained with the homologous RIA. Appl ication of the heterologous assay to dogs of different age revealed th at young dogs (3 months old) had 15-fold higher serum osteocalcin leve l than adult (>2 years old) dogs. In summary, we have (1) purified dog osteocalcin; (2) produced an antiserum against it; and (3) developed a heterologous RIA that could accurately measure dog osteocalcin, and could be used routinely to measure dog osteocalcin.