Vieira A
Department of Biochemistry, National Public Health Institute-KTL, Helsinki, Finland.
Mol Biotechnol. 1998 Dec;10(3):247-50. doi: 10.1007/BF02740845.
A simple, nonradioactive method is presented that can be used for performing large numbers of binding assays of cell membrane receptors with their ligands. The method adopts the simple membrane preparation and biotin-based quantitation methods of the semi-intact cell endocytosis assays. After binding of the biotinylated ligand to its receptors on the semi-intact cell membranes, a rapid centrifugation step separates the membranes from unbound ligand. Bound ligand is subsequently released by detergent, captured by a specific antibody coated on teh surface of microwells, and quantitated with peroxidase-conjugated streptavidin in a colorimetric assay. Using this assay, Scatchard analysis was performed on the data for the specific binding of iron-loaded transferrin to its receptors on mouse fibroblasts and yielded Kd values similar to those obtained with other published methods. The assay is sensitive, rapid, and also convenient, because aliquots of semi-intact cells can be stored frozen. The perforated plasma membrane of the cells offers the additional possibility of screening factors that interact with the cytoplasmic domain of the receptors for their possible effects on the parameters of the extracellular ligand-receptor interaction.
本文介绍了一种简单的非放射性方法，可用于大量进行细胞膜受体与其配体的结合测定。该方法采用了半完整细胞内吞测定法中简单的膜制备和基于生物素的定量方法。生物素化配体与半完整细胞膜上的受体结合后，通过快速离心步骤将膜与未结合的配体分离。随后，结合的配体用去污剂释放，被包被在微孔表面的特异性抗体捕获，并在比色测定中用过氧化物酶偶联的链霉亲和素进行定量。使用该测定法，对铁负载转铁蛋白与其在小鼠成纤维细胞上的受体的特异性结合数据进行了Scatchard分析，得到的解离常数（Kd）值与其他已发表方法获得的值相似。该测定法灵敏、快速且方便，因为半完整细胞的等分试样可以冷冻保存。细胞的穿孔质膜提供了额外的可能性，即筛选与受体胞质结构域相互作用的因子，以研究它们对细胞外配体-受体相互作用参数的可能影响。
