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.