Development and characterization of a polyclonal antiserum‐based radioimmunoassay for dog osteocalcin

Paolo Fanti, Giovanna Colombo, Chinghua Yao, Stephen A. Brown, Michael W. Vernon, Hartmut H. Malluche

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Determination of the serum concentration of the protein osteocalcin (OC) is useful for the noninvasive evaluation of bone metabolism. Because 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 of the concentration of this protein in dog serum. The antiserum expresses higher affinity for Ca2+‐bound than for Ca2+‐free OC (B50 at 10−5 versus 2 × 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 competitive binding of the peptide comprising amino acids 20–44 but not of other fragments; this suggests that the antigenic epitope of dog OC is located in the midmolecular region of the protein. The RIA displays excellent 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 antiserum displays limited species specificity since it cross‐reacts with human OC, but not with the protein from rodents. Consistent with previous observations in other in vivo models, the serum concentration of OC in experimental dogs is decreased significantly 7–10 days after thyroparathyroidectomy and it is unchanged 1 month following ovariohysterectomy.

Original languageEnglish (US)
Pages (from-to)745-752
Number of pages8
JournalJournal of Bone and Mineral Research
Volume8
Issue number6
DOIs
StatePublished - Jun 1993

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Orthopedics and Sports Medicine

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