De León D D, Asmerom Y
Department of Physiology, Loma Linda University School of Medicine, CA 92350, USA.
Endocrinology. 1997 May;138(5):2199-202. doi: 10.1210/endo.138.5.5237.
A chemiluminescent dot blot assay has been developed by our laboratory for rapid determinations of IGF-I in serum-free conditioned media (CM) collected from cultured cells. In contrast to IGF-I radioimmunoassays (RIAs), the IGF binding proteins (IGFBPs) did not interfere with the dot blot assay and did not require the laborious (and sometimes ineffective) removal of IGFBPs. Although all six IGFBPs were shown to bind to 125I IGF-I, none interfered with IGF-I detection on nitrocellulose dot blots. In contrast, an RIA using the same Oncogene monoclonal antibody (clone 82-9A) showed interference by IGFBP-1, IGFBP-2, and IGFBP-4. The IGF-I dot blot assay was sensitive (0.125-8.0 ng IGF-I), specific (assay crossreactivity with IGF-II is less than 1%), and reproducible (intra-assay variance < or = 6%; inter-assay variance < 12%) when chemiluminescence was quantified by phosphorimager and Molecular Analyst software (BioRad). The apparent sensitivity of the enhanced chemiluminescence (ECL) reagent to serum, precludes the use of this assay for IGF-I determination in serum or in serum-containing media.
我们实验室开发了一种化学发光斑点印迹法，用于快速测定从培养细胞中收集的无血清条件培养基（CM）中的IGF-I。与IGF-I放射免疫分析（RIA）不同，IGF结合蛋白（IGFBP）不会干扰斑点印迹法，也不需要费力地（有时还无效地）去除IGFBP。尽管所有六种IGFBP都显示能与125I IGF-I结合，但在硝酸纤维素斑点印迹上均不会干扰IGF-I的检测。相比之下，使用相同癌基因单克隆抗体（克隆82-9A）的RIA显示受到IGFBP-1、IGFBP-2和IGFBP-4的干扰。当通过磷光成像仪和Molecular Analyst软件（伯乐公司）对化学发光进行定量时，IGF-I斑点印迹法灵敏（0.125 - 8.0 ng IGF-I）、特异（与IGF-II的分析交叉反应性小于1%）且可重复（批内差异≤6%；批间差异<12%）。增强化学发光（ECL）试剂对血清的明显敏感性，使得该方法无法用于血清或含血清培养基中IGF-I的测定。
