一种用于测量胰岛素可调节葡萄糖转运蛋白的竞争性酶联免疫吸附测定法的验证

Validation of a competitive enzyme-linked immunosorbent assay for measuring the insulin-regulatable glucose transporter.

作者信息

Li S H, McNeill J H

机构信息

Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, British Columbia, Canada V6T 1Z3.

出版信息

J Pharmacol Toxicol Methods. 2000 Jan-Feb;43(1):85-90. doi: 10.1016/s1056-8719(00)00090-3.

DOI:10.1016/s1056-8719(00)00090-3

PMID:11091134

Abstract

A novel competitive enzyme-linked immunosorbent assay (ELISA) for measuring the insulin-regulatable glucose transporter (GLUT4) has been developed in our laboratory. The purpose of the present study was to verify the competitive ELISA assay. Towards this goal, a time-course study was conducted in control Wistar rat hearts to determine the points at which insulin stimulated an initial and a maximal GLUT4 translocation. Plasma and intracellular membrane fractions were purified from heart ventricles isolated from rats either in the basal state or injected with insulin. GLUT4 content in the membrane fractions was quantified with both the competitive ELISA method and also with enhanced chemiluminescence (ECL) Western blot. It was shown that after insulin injection plasma membrane GLUT4 level increased about 60% at 15 min and intracellular GLUT4 decreased about 40-50% at 5 min and remained at this level throughout the remaining 25 min by both methods. In conclusion, the data from this study demonstrate that the ELISA assay is reliable as verified by the Western blot method.

摘要

我们实验室已开发出一种用于检测胰岛素调节性葡萄糖转运蛋白（GLUT4）的新型竞争性酶联免疫吸附测定（ELISA）方法。本研究的目的是验证该竞争性ELISA检测方法。为实现这一目标，在对照Wistar大鼠心脏中进行了一项时间进程研究，以确定胰岛素刺激GLUT4发生初始转运和最大转运的时间点。从处于基础状态或注射胰岛素的大鼠分离出的心室中纯化血浆和细胞内膜组分。使用竞争性ELISA方法以及增强化学发光（ECL）免疫印迹法对膜组分中的GLUT4含量进行定量。结果显示，注射胰岛素后，两种方法均表明，在15分钟时质膜GLUT4水平增加约60%，在5分钟时细胞内GLUT4减少约40 - 50%，并在随后的25分钟内维持在该水平。总之，本研究数据表明，ELISA检测方法经免疫印迹法验证是可靠的。