Honda Y, Landale E C, Strong D D, Baylink D J, Mohan S
Department of Medicine, Loma Linda University, California 92357, USA.
J Clin Endocrinol Metab. 1996 Apr;81(4):1389-96. doi: 10.1210/jcem.81.4.8636339.
Insulin-like growth factor-binding protein-4 (IGFBP-4), like the five other IGFBPs present in human serum, acts as a transport protein for insulin-like growth factor I (IGF-I) and IGF-II and modulates their biological effects. To investigate the role of IGFBP-4 in the physiology of the IGF system, we developed a sensitive RIA for IGFBP-4 employing, as antigen, tracer, and standard, recombinant human IGFBP-4 (rhIGFBP-4) expressed in Escherichia coli as a fusion protein with glutathione S-transferase and affinity purified with glutathione-derivatized resin. Antibody against the rhIGFBP-4 fusion protein was raised in guinea pigs; tracer and standard were provided by the rhIGFBP-4 moiety that had been cleaved from the rhIGFBP-4 fusion protein and repurified by reverse phase high pressure liquid chromatography. We report that both IGFBP-4 purified from PC3 human prostate cell-conditioned medium and rhIGFBP-4 bound IGF and migrated in electrophoresis gels in an identical manner; that in gel permeation chromatography, rhIGFBP-4 coeluted with the IGFBP-4 present in human serum; and that both are equally immunoreactive with the IGFBP-4 antiserum. Employing this IGFBP-4 RIA, we determined that no IGFBP other than IGFBP-4 reacted with the IGFBP-4 antiserum, and that recovery of IGFBP-4 from serum samples exceeded 90% when exogenous IGFBP-4 was added and was unaffected by the addition of IGFs or by repeated freezing and thawing of the sample. We employed this IGFBP-4 RIA to demonstrate an increase in IGFBP-4 in TE85 human osteosarcoma cell-conditioned medium after treatment with dibutyryl cAMP, PTH, and 1,25-dihydroxyvitamin D3, agents known to increase the IGFBP-4 messenger ribonucleic acid level. Application of this RIA to the measurement of IGFBP-4 in human serum revealed that the circulating level of IGFBP-4 in 41 individuals in the 61-87 yr age group (546 +/- 135 microgram/L) was 35% higher than that in 24 individuals in the 23-40 yr age group (404 +/- 156 microgram/L). The mean circulating level of PTH was also 20% higher in the 61-87 yr group compared to that in the 23-40 yr group (P < 0.01). In addition, serum IGFBP-4 amounts showed a significant positive correlation with age (r = 0.54; P < 0.001) and serum PTH (r = 0.26; P < 0.01). These data validate this IGFBP-4 RIA and illustrate its utility in illuminating the physiological mechanisms that regulate IGFBP-4 in vivo and influence its effects on the IGFs in both normal and abnormal pathology and in aging.
胰岛素样生长因子结合蛋白4（IGFBP-4）与人类血清中存在的其他五种IGFBP一样，作为胰岛素样生长因子I（IGF-I）和IGF-II的转运蛋白，并调节它们的生物学效应。为了研究IGFBP-4在IGF系统生理学中的作用，我们开发了一种灵敏的IGFBP-4放射免疫分析法（RIA），该方法使用在大肠杆菌中表达的重组人IGFBP-4（rhIGFBP-4）作为抗原、示踪剂和标准品，rhIGFBP-4是与谷胱甘肽S-转移酶融合表达的，并经谷胱甘肽衍生化树脂亲和纯化。用豚鼠制备抗rhIGFBP-4融合蛋白的抗体；示踪剂和标准品由从rhIGFBP-4融合蛋白上切割下来并经反相高压液相色谱法重新纯化的rhIGFBP-4部分提供。我们报告，从PC3人前列腺细胞条件培养基中纯化的IGFBP-4和rhIGFBP-4都能结合IGF，并以相同的方式在电泳凝胶中迁移；在凝胶渗透色谱中，rhIGFBP-4与人血清中存在的IGFBP-4共同洗脱；并且两者与IGFBP-4抗血清的免疫反应性相同。使用这种IGFBP-4 RIA，我们确定除IGFBP-4外没有其他IGFBP与IGFBP-4抗血清反应，当添加外源性IGFBP-4时，血清样品中IGFBP-4的回收率超过90%，并且不受添加IGF或样品反复冻融的影响。我们使用这种IGFBP-4 RIA来证明用二丁酰环磷腺苷、甲状旁腺激素（PTH）和1,25-二羟基维生素D3处理后，TE85人骨肉瘤细胞条件培养基中IGFBP-4增加，已知这些试剂可增加IGFBP-4信使核糖核酸水平。将这种RIA应用于测量人血清中的IGFBP-4，结果显示61 - 87岁年龄组的41名个体中IGFBP-4的循环水平（546±135μg/L）比23 - 40岁年龄组的24名个体中（404±156μg/L）高35%。61 - 87岁组的PTH平均循环水平也比23 - 40岁组高20%（P<0.01）。此外，血清IGFBP-4含量与年龄（r = 0.54；P<0.001）和血清PTH（r = 0.26；P<0.01）呈显著正相关。这些数据验证了这种IGFBP-4 RIA，并说明了其在阐明体内调节IGFBP-4的生理机制以及在正常和异常病理及衰老过程中影响其对IGF作用方面的实用性。
