Premium
Development and characterization of a polyclonal antiserum‐based radioimmunoassay for dog osteocalcin
Author(s) -
Fanti Paolo,
Colombo Giovanna,
Yao Chinghua,
Brown Stephen A.,
Ver Michael W.,
Malluche Hartmut H.
Publication year - 1993
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650080613
Subject(s) - radioimmunoassay , polyclonal antibodies , antiserum , osteocalcin , endocrinology , medicine , chemistry , antibody , biology , immunology , biochemistry , alkaline phosphatase , enzyme
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 Ca 2+ ‐bound than for Ca 2+ ‐free OC (B 50 at 10 −5 versus 2 × 10 −4 dilution). Also, in the presence of Ca 2+ affinity is higher for the carboxylated than for the decarboxylated form of the protein, and under Ca 2+ ‐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.