Leung K C, Millard W J, Peters E, Markus I, Baumbach W R, Barnard R, Ho K K
Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales, Australia.
Endocrinology. 1995 Feb;136(2):379-85. doi: 10.1210/endo.136.2.7835268.
We have developed a ligand immunofunctional assay (LIFA) for quantifying the circulating functional GH-binding protein (GHBP) in the rat. This two-site solid-phase assay uses a capture monoclonal antibody (4.3) specific to the hydrophilic C-terminal segment of rat GHBP (rGHBP), saturation of binding with human GH, and a detection system of rabbit antihuman GH polyclonal antibody and peroxidase-conjugated antirabbit immunoglobulin G antibody. Results were compared with Scatchard estimates derived by immuno-precipitation with monoclonal antibody 4.3. This assay was used to determine the GHBP levels in male and female rats and to investigate the diurnal properties and dynamics of GH and GHBP interaction in 15-min blood sampling over a 6-h period. The dynamic range of the rLIFA was 0.15-20.0 nM recombinant rGHBP, with intraassay and interassay coefficients of variation of 10.5% (n = 20) and 12.9% (n = 12), respectively. Serum GHBP levels determined by the rLIFA and those derived from Scatchard estimates were strongly correlated (n = 8; beta = 0.55; r2 = 0.89; P = 0.0005). Male rats had lower GHBP levels (6.5 +/- 0.7 nM; mean +/- SE; n = 14) than female rats (35.4 +/- 2.7 nM; n = 15; P = 0.0001). In the diurnal study, male rats had higher GH peaks (312.5 +/- 121.6 ng/ml; n = 7) than female rats (96.5 +/- 15.4 ng/ml; n = 9; P < 0.0001). In contrast to the pulsatile secretion of GH, GHBP levels in both sexes remained stable and showed no relationship to secretory pulses of GH. However, the GH bursts significantly altered the distribution of the GH-GHBP complex in male rats. By saturation and mass analysis, the greater GH pulsatile secretion in male rats resulted in occupancy of GHBP from less than 5% at nadir to about 80% at secretory peaks, in contrast to the less than 5-15% range of GHBP occupancy in female rats. In male rats, greater than 80% of GH at secretory peaks existed in the free form, whereas in female rats, 16-23% of GH existed in the free form during pulsatile secretion. In summary, the rLIFA shows good correlation to Scatchard analysis using an identical antibody. We conclude that this assay provides a rapid, sensitive, and accurate measurement of the circulating functional GHBP in the rat, and that it facilitates the study of GH and GHBP dynamics under a range of physiological conditions.
我们已开发出一种配体免疫功能测定法（LIFA），用于定量大鼠体内循环的功能性生长激素结合蛋白（GHBP）。这种双位点固相测定法使用针对大鼠GHBP（rGHBP）亲水性C末端片段的捕获单克隆抗体（4.3），用人GH使结合饱和，以及兔抗人GH多克隆抗体和过氧化物酶偶联的抗兔免疫球蛋白G抗体的检测系统。将结果与用单克隆抗体4.3免疫沉淀得出的Scatchard估计值进行比较。该测定法用于测定雄性和雌性大鼠的GHBP水平，并在6小时内每15分钟采集一次血液样本，研究GH和GHBP相互作用的昼夜特性及动态变化。rLIFA的动态范围为0.15 - 20.0 nM重组rGHBP，批内和批间变异系数分别为10.5%（n = 20）和12.9%（n = 12）。通过rLIFA测定的血清GHBP水平与从Scatchard估计值得出的水平高度相关（n = 8；β = 0.55；r2 = 0.89；P = 0.0005）。雄性大鼠的GHBP水平（6.5±0.7 nM；平均值±标准误；n = 14）低于雌性大鼠（35.4±2.7 nM；n = 15；P = 0.0001）。在昼夜研究中，雄性大鼠的GH峰值（312.5±121.6 ng/ml；n = 7）高于雌性大鼠（96.5±15.4 ng/ml；n = 9；P < 0.0001）。与GH的脉冲式分泌不同，两性的GHBP水平保持稳定，且与GH的分泌脉冲无关。然而，GH脉冲显著改变了雄性大鼠中GH - GHBP复合物的分布。通过饱和分析和质量分析，雄性大鼠中更大的GH脉冲式分泌导致GHBP的占有率从最低点时的不到5%增加到分泌峰值时的约80%，而雌性大鼠中GHBP的占有率范围为不到5 - 15%。在雄性大鼠中，分泌峰值时超过80%的GH以游离形式存在，而在雌性大鼠中，脉冲式分泌期间16 - 23%的GH以游离形式存在。总之，rLIFA与使用相同抗体的Scatchard分析显示出良好的相关性。我们得出结论，该测定法能快速、灵敏且准确地测量大鼠体内循环的功能性GHBP，并且有助于在一系列生理条件下研究GH和GHBP的动态变化。
