DEVELOPMENT AND CHARACTERIZATION OF A POLYCLONAL ANTISERUM-BASED RADIOIMMUNOASSAY FOR DOG OSTEOCALCIN

Determination of the serum concentration of the protein osteocalcin (O C) is useful for the noninvasive evaluation of bone metabolism. Becaus e the dog is an excellent experimental model for the study of bone, we produced and characterized a polyclonal antiserum specific for dog OC and used it to develop a radioimmunoassay (RIA) for the measurement o f the concentration of this protein in dog serum. The antiserum expres ses higher affinity for Ca2+-bound than for Ca2+-free OC (B50 at 10(-5 ) versus 2 x 10(-4) dilution). Also, in the presence of Ca2+ affinity is higher for the carboxylated than for the decarboxylated form of the protein, and under Ca2+-free conditions the affinity is equal for the two forms. The study of peptide fragments of OC demonstrates competit ive binding of the peptide comprising amino acids 20-44 but not of oth er fragments; this suggests that the antigenic epitope of dog OC is lo cated in the midmolecular region of the protein. The RIA displays exce llent sensitivity for the measurement of OC in blood (detection limit 0.31 ng/ml), with intraassay and interassay variations of 4.6 and 6.8% , respectively. Analysis of gel chromatography fractions of normal dog serum shows that greater than 90% of the antigenic material coelutes with purified radiolabeled dog OC. Test of parallelism reveals lack of interference of serum constituents with the binding assay. The antise rum displays limited species specificity since it cross-reacts with hu man OC, but not with the protein from rodents. Consistent with previou s observations in other in vivo models, the serum concentration of OC in experimental dogs is decreased significantly 7-10 days after thyrop arathyroidectomy and it is unchanged 1 month following ovariohysterect omy.