Fanti P, Colombo G, Yao C, Brown S A, Vernon M W, Malluche H H
Division of Nephrology, Bone and Mineral Metabolism, University of Kentucky, Lexington.
J Bone Miner Res. 1993 Jun;8(6):745-52. doi: 10.1002/jbmr.5650080613.
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 (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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
测定血清骨钙素（OC）浓度有助于对骨代谢进行无创评估。由于犬是研究骨骼的优良实验模型，我们制备了针对犬OC的多克隆抗血清并对其特性进行了鉴定，并用它开发了一种放射免疫分析法（RIA）来测量犬血清中该蛋白的浓度。该抗血清对结合钙的OC的亲和力高于对游离钙OC的亲和力（B50分别为10^(-5)和2×10^(-4)稀释度）。此外，在有钙离子存在的情况下，该抗血清对羧化形式蛋白的亲和力高于对脱羧形式蛋白的亲和力；而在无钙离子条件下，两种形式蛋白的亲和力相同。对OC肽片段的研究表明，包含氨基酸20 - 44的肽段具有竞争性结合，而其他片段则没有；这表明犬OC的抗原表位位于该蛋白的分子中部区域。该RIA对血液中OC的测量具有出色的灵敏度（检测限为0.31 ng/ml），批内和批间变异分别为4.6%和6.8%。对正常犬血清凝胶色谱级分的分析表明，超过90%的抗原物质与纯化的放射性标记犬OC共洗脱。平行性测试表明血清成分对结合测定没有干扰。该抗血清的种属特异性有限，因为它与人OC发生交叉反应，但不与啮齿动物的蛋白发生交叉反应。（摘要截短至250字）
