De León D D, Terry C, Nissley S P
National Institutes of Health, National Cancer Institute, Metabolism Branch, Bethesda, MD 20892.
Endocrinology. 1994 Apr;134(4):1960-3. doi: 10.1210/endo.134.4.7511096.
A dot blot method for the detection of picogram quantities of human and rat insulin-like growth factor II (IGF-II) in serum-free conditioned media is described. The crossreactivity of human recombinant IGF-I in the assay was < 10%. None of the IGF binding proteins (IGFBP 1-6) diminished the IGF-II signal. In contrast, significant interference by the IGFBPs was observed when the same concentrations of IGFBPs and 125I-IGF-II were used in a radioimmunoassay which utilized the same antibody. Why IGF-II is detected in the dot blot assay without IGFBP interference is not understood. We speculate that the conformation of the IGF-II/binding protein complex may be altered by binding to the nitrocellulose, exposing the IGF-II epitope that is recognized by the antibody. IGF-II was detected in 1 microliter of serum-free conditioned media from BRL 3A cells (which secrete IGF-II) while no signal was generated by 50 microliters of BRL 3A2 conditioned media (which do not secrete IGF-II). In summary, this method is ideal for screening cells in serum free-culture for production of IGF-II without the need for separation of IGF-II from cell derived IGFBPs.
本文描述了一种斑点印迹法，用于检测无血清条件培养基中皮克级量的人和大鼠胰岛素样生长因子II（IGF-II）。在该检测中，人重组IGF-I的交叉反应性<10%。IGF结合蛋白（IGFBP 1-6）均未减弱IGF-II信号。相比之下，当在使用相同抗体的放射免疫分析中使用相同浓度的IGFBP和125I-IGF-II时，观察到IGFBP有显著干扰。为何在斑点印迹分析中能检测到无IGFBP干扰的IGF-II尚不清楚。我们推测，IGF-II/结合蛋白复合物的构象可能因与硝酸纤维素结合而改变，从而暴露出被抗体识别的IGF-II表位。在来自BRL 3A细胞（分泌IGF-II）的1微升无血清条件培养基中检测到了IGF-II，而50微升BRL 3A2条件培养基（不分泌IGF-II）未产生信号。总之，该方法非常适合在无血清培养中筛选产生IGF-II的细胞，无需从细胞来源的IGFBP中分离IGF-II。
