A HIGHLY PRECISE REPORTER GENE BIOASSAY FOR TYPE-I INTERFERON

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 pla smid 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 indu cibility by IFN-beta. A linear dose-response relationship was found be tween 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 includ ing IFN-gamma could be detected. In order to render the IFN-beta repor ter gene assay protocol more suitable for routine assays, a 3 x 3 bala nced parallel line assay design was applied using a 96-well luminomete r for luminescence measurement. The assay was shown to be precise with a coefficient of variation of less than 9%. This assay is characteriz ed by high precision coupled to high efficiency, as reflected by a ver y short assay duration (1 day), when compared to the classical cytopat hic effect assays for IFNs and the previously published IFN reporter g ene assay based on growth hormone measurement (Lleonart, R., Naf, D., Browning, H. and Weissmann, C. (1990) A never, quantitative bioassay f or type I interferon using a recombinant indicator cell line. Biotechn ology 8, 1263-1267).