Carlsson L M, Rowland A M, Clark R G, Gesundheit N, Wong W L
Department of Immunology Research, Genentech, Inc., South San Francisco, California 94080.
J Clin Endocrinol Metab. 1991 Dec;73(6):1216-23. doi: 10.1210/jcem-73-6-1216.
Human serum contains a high affinity GH-binding protein (GHBP) whose amino-terminal sequence is identical to the extracellular domain of the GH receptor. Current methods that measure GHBP are laborious, require size or charcoal separation of the GH/GHBP complex, and may be influenced by ambient GH concentrations. We have developed a novel assay method that allows quantitation of the total amount of functional GHBP in serum or plasma. The assay can also be used to measure the concentration of the circulating GH/GHBP complex. An anti-GHBP monoclonal antibody, which recognizes both free GHBP and GH-bound GHBP, is used to capture the GHBP on a microtiter plate. Recombinant human GH is added to saturate all binding sites, and an anti-GH antibody conjugated with horseradish peroxidase is used to detect the amount of GH (endogenous and exogenous) bound to the GHBP. The same procedure, but without incubation with GH, allows measurement of the endogenous GH/GHBP complex. The assay is sensitive (detection range, 31-2000 pmol/L), with average inter- and intraassay precisions of 11.3% and 7.3%, respectively. Measurements in random blood samples from 16 healthy adults showed that all subjects had clearly detectable GHBP concentrations (range, 65.8-305.6 pmol/L). In contrast, GHBP levels were undetectable in samples from 2 patients with Laron-type dwarfism. We believe that this ligand-mediated immunofunctional assay, which combines the simplicity and specificity of an enzyme-linked immunosorbent assay with the ability to detect only biochemically active binding protein, will be useful for studies of the role of the GHBP in health and disease.
人血清中含有一种高亲和力生长激素结合蛋白（GHBP），其氨基末端序列与生长激素受体的细胞外结构域相同。目前测量GHBP的方法繁琐，需要对生长激素/生长激素结合蛋白复合物进行大小分离或用活性炭分离，且可能受环境生长激素浓度的影响。我们开发了一种新的检测方法，可对血清或血浆中功能性GHBP的总量进行定量。该检测方法还可用于测量循环中的生长激素/生长激素结合蛋白复合物的浓度。一种抗GHBP单克隆抗体，可识别游离的GHBP和与生长激素结合的GHBP，用于在微量滴定板上捕获GHBP。加入重组人生长激素以饱和所有结合位点，并用与辣根过氧化物酶偶联的抗生长激素抗体检测与GHBP结合的生长激素（内源性和外源性）的量。同样的步骤，但不与生长激素孵育，可测量内源性生长激素/生长激素结合蛋白复合物。该检测方法灵敏（检测范围为31 - 2000 pmol/L），平均批间和批内精密度分别为11.3%和7.3%。对16名健康成年人随机血样的检测表明，所有受试者的GHBP浓度均清晰可测（范围为65.8 - 305.6 pmol/L）。相比之下，2例拉伦型侏儒症患者的样本中未检测到GHBP水平。我们认为，这种配体介导的免疫功能检测方法，将酶联免疫吸附测定的简单性和特异性与仅检测生化活性结合蛋白的能力相结合，将有助于研究GHBP在健康和疾病中的作用。
