Tar A, Hocquette J F, Souberbielle J C, Clot J P, Brauner R, Postel-Vinay M C
Unité de Biologie et Pathologie de la Croissance et du Développement, INSERM Unité 30, Hôpital des Enfants-Malades, Paris, France.
J Clin Endocrinol Metab. 1990 Nov;71(5):1202-7. doi: 10.1210/jcem-71-5-1202.
A technique using high pressure liquid chromatography gel filtration was used to evaluate GH-binding proteins (BP) in human plasma; eluate was monitored for radioactivity in a gamma-detection system connected to a computer. Plasma (200 microL) was incubated with [125I]human (h) GH (200,000 cpm) at 4 C for 20 h. The main GH-BP (peak II) was well separated from free [125I]hGH (peak III) and from a higher mol wt complex (peak I), which was minor. In our control plasma, the specific binding of [125I]hGH to peak II BP (II-BP) was 32.2 +/- 0.6% of the radioactivity. Scatchard analyses indicate an association constant of 3.6-7.4 X 10(8) M-1 and a binding capacity ranging from 24-86 ng/mL for peak II-BP in five normal adult plasma samples. Peak I material, separated from plasma of boys with pubertal delay, bound hGH with a low affinity (3 x 10(6) M-1) and a very high capacity (2 micrograms/mL). In cross-linking experiments, peak I appeared as two proteins of 165 and 174 kD; these mol wt were much higher than that of peak II-BP, previously estimated at 53,000. hGH complexed to peak II-BP remained fully immunoreactive with use of the anti-hGH antibodies of our assay. In plasma containing 10-20 micrograms/L hGH, the proportion of bound hormone (peak II) was 44.5 +/- 2.3%, whereas the amount of hGH in peak I was very low or undetectable. Specific binding of hGH to II-BP was lowest during the first year of life and highest in adulthood. No sex difference was found. I-BP is differentially regulated, since its binding activity was significantly lower in adults than in prepubertal children. Normal values for age should be taken into account to interpret GH-binding activity, particularly in children 2 yr of age or younger. Our GH binding assay offers important gains in terms of rapidity and resolution; it has permitted a clear separation and characterization of the two GH-binding components present in human plasma.
采用高压液相色谱凝胶过滤技术评估人血浆中的生长激素结合蛋白（BP）；洗脱液在连接计算机的γ检测系统中监测放射性。将血浆（200微升）与[125I]人（h）生长激素（200,000计数/分钟）在4℃孵育20小时。主要的生长激素结合蛋白（峰II）与游离的[125I]hGH（峰III）以及一种较高分子量的复合物（峰I，含量较少）能很好地分离。在我们的对照血浆中，[125I]hGH与峰II BP（II-BP）的特异性结合为放射性的32.2±0.6%。Scatchard分析表明，在五个正常成人血浆样本中，峰II-BP的缔合常数为3.6 - 7.4×10⁸ M⁻¹，结合容量为24 - 86纳克/毫升。从青春期延迟男孩的血浆中分离出的峰I物质，以低亲和力（3×10⁶ M⁻¹）和非常高的容量（2微克/毫升）结合hGH。在交联实验中，峰I表现为两种分子量分别为165和174千道尔顿的蛋白质；这些分子量远高于先前估计的峰II-BP的分子量53,000。与峰II-BP结合的hGH在我们的检测中使用抗hGH抗体时仍保持完全免疫反应性。在含有10 - 20微克/升hGH的血浆中，结合激素（峰II）的比例为44.5±2.3%，而峰I中的hGH量非常低或无法检测到。hGH与II-BP的特异性结合在生命的第一年最低，在成年期最高。未发现性别差异。I-BP受到不同调节，因为其结合活性在成年人中明显低于青春期前儿童。在解释生长激素结合活性时，应考虑年龄的正常值，特别是在2岁及以下的儿童中。我们的生长激素结合检测在速度和分辨率方面有重要提升；它能够清晰地分离和鉴定人血浆中存在的两种生长激素结合成分。
