Wiener H L, Reith M E
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John's University, Jamaica, New York 11439.
Anal Biochem. 1992 Nov 15;207(1):58-62. doi: 10.1016/0003-2697(92)90499-w.
Radioligand binding assays are routinely utilized in laboratories throughout the world to study receptors and their related binding sites, carrier proteins, and enzymes. To accurately estimate equilibrium binding parameters, such as the equilibrium dissociation constant (Kd) and maximal number of binding sites (Bmax), the investigator must know the correct value of the specific activity of the radioligand. If the specific activity is overestimated the Kd and Bmax values will be underestimated, while underestimation of the specific activity results in an overestimation of the Kd and Bmax. The present communication describes a simple and rapid method for determining the specific activity of a radioligand using homologous competition binding assays. Performing the competition assays at two or more different concentrations of the radioligand allows the specific activity to be determined from the IC50 values without the need of analytical methods to quantify minute amounts of the radioligand. In addition to providing the specific activity, use of this method estimates the Kd for the radioligand. This method was utilized to determine the specific activity and Kd for two blockers of the dopamine uptake carrier, [3H]GBR-12935 and [3H]-CFT, which share a common binding site in the striatum.
放射性配体结合测定法在世界各地的实验室中被常规用于研究受体及其相关结合位点、载体蛋白和酶。为了准确估计平衡结合参数，如平衡解离常数（Kd）和最大结合位点数（Bmax），研究者必须知道放射性配体比活度的正确值。如果比活度被高估，Kd和Bmax值将被低估，而比活度被低估则会导致Kd和Bmax被高估。本通讯描述了一种使用同源竞争结合测定法测定放射性配体比活度的简单快速方法。在两种或更多不同浓度的放射性配体下进行竞争测定，可以从IC50值确定比活度，而无需使用分析方法来定量微量的放射性配体。除了提供比活度外，使用该方法还可以估计放射性配体的Kd。该方法用于测定两种多巴胺摄取载体阻滞剂[3H]GBR - 12935和[3H]-CFT的比活度和Kd，它们在纹状体中共享一个共同的结合位点。
