Validation of a HeLa <i>Mx2</i>/Luc Reporter Cell Line for the Quantification of Human Type I Interferons

Abstract

Although antiviral assays have been the most widely available biological assays for interferons (IFNs), they are less sensitive and provide considerable interassay variation. In this study, we demonstrate a new reporter cell line, which is based on HeLa cells transfected with a plasmid containing a human Mx2 promoter driving a luciferase (Luc) cDNA. To characterize the specific gene expression profiles induced by interferon alpha, we analyzed the microarray results of interferon response gene expression induced by IFN-α2a or IFN-α2b treatment with HeLa cells. We found that the Mx2 gene increased the most by treatment with both IFN-α2a and IFN-α2b. Based on this result, we designed a reporter cell line, HeLa-Mx2, suitable for determination of IFN-α. HeLa cells were stably transfected with the luciferase gene under the control of Mx2 promoter. The expression of luciferase can be easily measured for luminescence using a 96-well luminometer and has been correlated with the concentration of added IFN and cell density. In the validation results, our reporter cell line had specificity for type I IFN, but the significant effects of a number of other cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, IL-2, IL-5, IL-6 and GM-CSF, or type II interferon (IFN-γ) were not observed. Moreover, the robustness of our cell line is demonstrated by the lack of an effect of the HeLa-Mx2 cell culture’s age on the performance of the reporter gene assay. The reporter gene assay demonstrated reproducible dose-response curves for IFN-α2a in the range of 1–10,000 IU/ml. The 95% confidential limit and total coefficient of variation estimates ranged between 96 and 116 and 10.51% in the reducible range mentioned above, respectively. In conclusion, we established a stable IFN-responsible HeLa-Mx2 cell line, which has advantages with regard to simplicity, selectivity, and reliability over conventional cytopathic effect reduction assays used to quantify IFN-α activity.

IFN Regulatory Factor 5 Is Required for Disease Development in the FcγRIIB−/−Yaa and FcγRIIB−/− Mouse Models of Systemic Lupus Erythematosus

“…We therefore measured mRNA expression of the type I IFN-regulated genes IFIT1 and MX2 (42, 43) in B220 + splenocytes and kidney from additional cohorts of Irf5 +/+ RII. Yaa, Irf5 −/− RII.…”

Detection of interferon alpha protein reveals differential levels and cellular sources in disease

“…However, despite almost 60 years of active research in this field, the direct measurement of type I IFN protein in biological samples has remained elusive. Type I IFN mRNA is usually present at only trace levels in PBMCs from healthy individuals, and current ELISAs have proven either insensitive or unreliable, leading to the development of proxy assays based on type I IFN signaling (Hua et al, 2006; Niewold et al, 2009; Seo et al, 2009; Li et al, 2010; Berger Rentsch and Zimmer, 2011). Such presumed low levels of circulating IFN protein likely reflect the high biological potency of these cytokines, with most cell types expressing a type I IFN receptor.…”

Bioactivity Determination of Native and Variant Forms of Therapeutic Interferons

“…These RGAs employed a variety of IFN responsive promoters including Mx, ISRE, GFAP [16–26]. Using Mx2/Luc reporter cell line, Seo et al [23] reported the RGA is even more sensitive than the ISRE/Luc reporter cell line. While previous studies have provided strong evidences that RGA is advantageous compared to the traditional AVA with respect to simplicity, rapidity and reproducibility, optimization and/or modification of the RGAs also appear to be necessary since some systems are associated with narrow detection range or require extra steps after the addition of IFN.…”