De Leenheer A P, Bauwens R M
Clin Chim Acta. 1985 Oct 31;152(1-2):143-54. doi: 10.1016/0009-8981(85)90185-8.
Human plasma and serum levels for 1 alpha,25-dihydroxyvitamin D were determined by a cytosol radioreceptor assay (RRA) and a radioimmunoassay (RIA). For both assays, 1.5 ml of human serum or plasma is used. Prior to RRA or RIA, extraction with benzene is performed followed by 'high-performance' liquid chromatography (HPLC) on a silica column (25 X 0.46 cm) with hexane/isopropanol (9/1 by vol), to isolate 1 alpha,25-dihydroxyvitamin D from the other vitamin D metabolites. The cytosol receptor was isolated from the intestine of healthy chickens. The antisera were raised in rabbits to 1 alpha,25-dihydroxyvitamin D3-3-hemisuccinate coupled to bovine serum albumin. The standard curves for RRA and RIA are prepared with 1 alpha,25-dihydroxyvitamin D3. 1 alpha,25-dihydroxy[3H]vitamin D3 of high spec act (158 kCi/mol) is used as tracer. The reactants are incubated for 16 h at 4 degrees C. Then, bound and free ligand are separated after the addition of dextran-coated charcoal. Both assays have a sensitivity of 2 pg/tube. The cytosol receptor and the antibodies have about the same absolute affinity for 1 alpha,25-dihydroxyvitamin D3 but the cytosol receptor has a higher relative affinity for 1 alpha,25-dihydroxyvitamin D3 (compared with other vitamin D metabolites). Reproducibility and precision are better for the RIA. The between- and within-assay CVs are 16.0% (mean = 58.7 ng/l, n = 16) and 11.2% (mean = 52.1 ng/l, n = 15), respectively, for RRA and 12.6% (mean = 61.8 ng/l, n = 27) and 7.4% (mean = 61.8 ng/l, n = 15), respectively using RIA. Reference values obtained by both assays on healthy males and healthy premenopausal females are the same for both sexes; 53.9 +/- 31.0 ng/l (n = 46) using RRA and 51.8 +/- 30.2 ng/l (n = 91) for RIA (mean +/- 2 SD).
采用胞质溶胶放射受体分析法（RRA）和放射免疫分析法（RIA）测定人血浆和血清中1α,25 - 二羟基维生素D的水平。两种分析方法均使用1.5毫升人血清或血浆。在进行RRA或RIA之前，先用苯萃取，然后在硅胶柱（25×0.46厘米）上用己烷/异丙醇（体积比9/1）进行“高效”液相色谱（HPLC），以从其他维生素D代谢产物中分离出1α,25 - 二羟基维生素D。胞质溶胶受体从健康鸡的肠道中分离得到。抗血清是在兔子体内针对与牛血清白蛋白偶联的1α,25 - 二羟基维生素D3 - 3 - 半琥珀酸酯产生的。RRA和RIA的标准曲线用1α,25 - 二羟基维生素D3绘制。使用高比活度（158 kCi/mol）的1α,25 - 二羟基[3H]维生素D3作为示踪剂。反应物在4℃下孵育16小时。然后，加入葡聚糖包被的活性炭后分离结合型和游离型配体。两种分析方法的灵敏度均为2 pg/管。胞质溶胶受体和抗体对1α,25 - 二羟基维生素D3的绝对亲和力大致相同，但胞质溶胶受体对1α,25 - 二羟基维生素D3的相对亲和力更高（与其他维生素D代谢产物相比）。RIA的重现性和精密度更好。RRA的批间和批内变异系数分别为16.0%（平均值 = 58.7 ng/l，n = 16）和11.2%（平均值 = 52.1 ng/l，n = 15），RIA的批间和批内变异系数分别为12.6%（平均值 = 61.8 ng/l，n = 27）和7.4%（平均值 = 61.8 ng/l，n = 15）。两种分析方法在健康男性和健康绝经前女性中获得的参考值在两性中相同；RRA为53.9±31.0 ng/l（n = 46），RIA为51.8±30.2 ng/l（n = 91）（平均值±2标准差）。
