一种用于检测干扰素调节的2-5A合成酶的灵敏测定法。

A sensitive assay for the IFN-regulated 2-5A synthetase enzyme.

作者信息

Hassel B A, Ts'o P O

机构信息

Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205.

出版信息

J Virol Methods. 1994 Dec;50(1-3):323-34. doi: 10.1016/0166-0934(94)90187-2.

DOI:10.1016/0166-0934(94)90187-2

PMID:7536199

Abstract

2-5A synthetase is the central enzyme of the 2-5A system, an important mediator of interferon action. An assay capable of detecting low, yet biologically important levels of 2-5A synthetase enzyme activity is described. The purification of enzyme reaction products on SepPak C-18 cartridges resulted in a significant reduction in background, when a high specific activity substrate was used to label the 2-5A. Quantitation of labeled 2-5A by chromatography and scintillation counting provided a means of detecting femptomolar amounts of 2-5A. The combination of these procedures accounts for a 3-4 log increase in sensitivity over existing assays. This degree of sensitivity should permit a more accurate determination of the 2-5A synthetase activity in vivo leading to a better understanding of the role of the 2-5A system in virus infection and other cellular processes.

摘要

2-5A合成酶是2-5A系统的核心酶，该系统是干扰素作用的重要介质。本文描述了一种能够检测低水平但具有生物学重要意义的2-5A合成酶活性的检测方法。当使用高比活性底物标记2-5A时，在SepPak C-18柱上对酶反应产物进行纯化可显著降低背景。通过色谱法和闪烁计数对标记的2-5A进行定量，提供了一种检测飞摩尔量2-5A的方法。这些方法的结合使灵敏度比现有检测方法提高了3至4个数量级。这种灵敏度水平应能更准确地测定体内2-5A合成酶的活性，从而更好地理解2-5A系统在病毒感染和其他细胞过程中的作用。