STUDIES ON THE MODE OF ACTION OF CALCIFEROL .13. DEVELOPMENT OF A RADIOIMMUNOASSAY FOR VITAMIN-D-DEPENDENT CHICK INTESTINAL CALCIUM-BINDING PROTEIN AND TISSUE DISTRIBUTION
STUDIES ON THE MODE OF ACTION OF CALCIFEROL .13. DEVELOPMENT OF A RADIOIMMUNOASSAY FOR VITAMIN-D-DEPENDENT CHICK INTESTINAL CALCIUM-BINDING PROTEIN AND TISSUE DISTRIBUTION
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DOI：
10.1210/endo-104-5-1495
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发表时间：
1979-01-01
期刊：
影响因子：
4.8
通讯作者：
NORMAN, AW
中科院分区：
文献类型：
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作者：
CHRISTAKOS, S;FRIEDLANDER, EJ;NORMAN, AW
建立了鸡肠道钙结合蛋白(CABP)的放射免疫分析方法，灵敏度为1 ng。用高纯度维生素D依赖的鸡肠CABP注射兔产生抗血清。该方法采用双抗体技术，用氯胺T制备~(125)I标记的CABP，与正常兔、大鼠和人血清蛋白无交叉反应。鸡肠道和肾脏CABP的RIA测定结果与放射免疫扩散法测定的CABP结果具有良好的相关性。该方法重现性好(批间变异16.3%)，精密度高(批内变异4.0%)。6.5nmol1，25-二羟基维生素D3给药后8h，鸡血清(2.7 ng/ml血清)中的免疫活性钙结合蛋白(ICaBP)浓度即可测得，20h时最高达11 ng/ml，鸡血清中钙结合蛋白的含量与维生素D3或1，25-二羟基维生素D3的剂量有关。维生素D缺乏雏鸡和脊椎病雏鸡各组织中的iCaBP浓度显著高于血清中的iCaBP浓度。从肾脏(480 ng/mg蛋白)和下丘脑(275 ng/mg蛋白)到心肌中几乎检测不到的水平(1.1 ng/mg蛋白)，脊椎病雏鸡的iCaBP的值范围很大。每天补充1.3nmol维生素D3 2wk后，iCaBP在十二指肠(25u.g/mg蛋白质)、空肠(32u.g/ml)、回肠(10u.g/mg)、肾脏(3.1u.g/mg)、胰腺(141 ng/mg)和骨骼(110 ng/mg)的水平最高。在其他几个组织中也检测到较低浓度的iCaBP。在除肝脏和骨骼肌以外的所有组织中，RIA中交叉反应的蛋白质在免疫化学上与高纯度的肠道CABP相似(通过平行免疫稀释曲线确定)。甲状旁腺iCaBP对维生素D及其代谢产物1，25-二羟基维生素D和24，25-二羟基维生素D3的反应模式与肠道CABP相似。骨骼中的iCaBP水平与饮食中钙和磷的变化呈相反的反应，肠道中的CaBP水平也是如此。通过使用RIA，可以定量测定鸡CABP的微量水平，并可以更有效地探测维生素D内分泌系统。
A RIA [radioimmunoassay] for chick intestinal Ca-binding protein (CaBP) was developed with a sensitivity of 1 ng. The antiserum was generated in rabbits injected with highly purified vitamin D-dependent chick intestinal CaBP. The assay employs the double antibody technique and 125I-labeled CaBP was prepared using chloramine T. Low MW peptide hormones and normal rabbit, rat and human serum proteins show no cross-reactivity in the assay. Measurements of chick intestinal and kidney CaBP by RIA showed a good correlation with measurements of CaBP by the radial immunodiffusion method. The assay is reproducible (interassay variability, 16.3%) and precise (intraasay variability, 4.0%). The concentration of immunoreactive CaBP (iCaBP) in chick serum (2.7 ng/ml serum) can be measured as early as 8 h after the administration of 6.5 nmol 1,25-dihydroxyvitamin D3; a maximum of 11 ng/ml is reached at 20 h. The level of CaBP in chick serum was dependent on the dose of vitamin D3 or 1,25-dihydroxyvitamin D3 administered to the animal. The concentration of iCaBP in various tissues of the vitamin D-replete and the rachitic chick was significantly higher than serum levels of iCaBP. The values for iCaBP in the rachitic chick ranged from a high value for the kidney (480 ng/mg protein) and hypothalamus (275 ng/mg protein) to a barely detectable level in the myocardium (1.1 ng/mg protein). After administering 1.3 nmol vitamin D3 daily for 2 wk, the level of iCaBP was highest in the duodenum (25 .mu.g/mg protein), jejunum (32 .mu.g/ml), ileum (10 .mu.g/mg), kidney (3.1 .mu.g/mg), pancreas (141 ng/mg) and bone (110 ng/mg). Lower concentrations of iCaBP were detected in several other tissues. In all tissues except liver and skeletal muscle, the protein crossreacting in the RIA was immunochemically similar (as determined by parallel immunodilution curves) to highly purified intestinal CaBP. iCaBP in parathyroid glands responded to vitamin D and its metabolites 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D3 in a qualitatively similar pattern to intestinal CaBP. Levels of iCaBP in bone responded inversely to changes in dietary Ca and P, as did the levels of CaBP in the intestine. By employing the RIA, minute levels of chick CaBP can be quantitated and the vitamin D endocrine system can be more effectively probed.