Canosi U, Mascia M, Gazza L, Serlupi-Crescenzi O, Donini S, Antonetti F, Galli G
Instituto di Ricerca Cesare Serono SpA, Rome, Italy.
J Immunol Methods. 1996 Nov 29;199(1):69-76. doi: 10.1016/s0022-1759(96)00168-8.
We describe the setting up and validation of a reporter gene assay for type I IFN based on monkey Vero cells transfected with pMx-Luc, a plasmid carrying the luciferase gene under the control of the type I IFN inducible mouse Mx1 promoter. Vero cells were stably transfected with pMx-Luc and clone 3-143/5 was selected on the basis of luciferase inducibility by IFN-beta. A linear dose-response relationship was found between 1 and 16 IU/ml IFN-beta. The assay was shown to be specific for IFN-alpha and -beta as no effect by a number of other cytokines including IFN-gamma could be detected. In order to render the IFN-beta reporter gene assay protocol more suitable for routine assays, a 3 x 3 balanced parallel line assay design was applied using a 96-well luminometer for luminescence measurement. The assay was shown to be precise with a coefficient of variation of less than 9%. This assay is characterized by high precision coupled to high efficiency, as reflected by a very short assay duration (1 day), when compared to the classical cytopathic effect assays for IFNs and the previously published IFN reporter gene assay based on growth hormone measurement (Lleonart, R., Näf, D., Browning, H. and Weissmann, C. (1990) A novel, quantitative bioassay for type 1 interferon using a recombinant indicator cell line. Biotechnology 8, 1263-1267).
我们描述了一种基于I型干扰素的报告基因检测方法的建立和验证，该方法使用用pMx-Luc转染的猴Vero细胞，pMx-Luc是一种携带萤光素酶基因的质粒，该基因受I型干扰素诱导的小鼠Mx1启动子控制。用pMx-Luc稳定转染Vero细胞，并根据β干扰素对萤光素酶的诱导性选择克隆3-143/5。在1至16 IU/ml的β干扰素之间发现了线性剂量反应关系。该检测方法对α干扰素和β干扰素具有特异性，因为未检测到包括γ干扰素在内的许多其他细胞因子的作用。为了使β干扰素报告基因检测方案更适合常规检测，采用了3×3平衡平行线检测设计，使用96孔发光计进行发光测量。该检测方法显示出精确性，变异系数小于9%。该检测方法的特点是高精度与高效率相结合，与经典的干扰素细胞病变效应检测方法以及先前发表的基于生长激素测量的干扰素报告基因检测方法相比，检测时间非常短（1天）。
