Measuring type I interferon using reporter gene assays based on readily available cell lines.

ABSTRACT

Cell lines stably transfected with genes responding to Type I interferons (IFN) are potentially a useful alternative to enzyme linked immuo-assays (ELISAs) or assays based on resistance of a test cell line to virus infection using cell death or infection endpoints. Increasingly available are a variety of commercial cell lines developed for reporter gene assays (RGAs) which are responsive to IFN exposure. These cells produce a soluble gene product which can be readily quantified using multiwell plate spectrophotometers or luminometers. We have investigated RAW-Blue ISG™ and B16-Blue IFNα/ß™ cells (InvivoGen) which produce secreted embryonic alkaline phosphatase (SEAP) as a RGA to measure Interferon alpha (IFNα) and beta (IFNß). These cells showed a log-linear response over 4 logs of IFN concentration between 10 and 100,000 Units/ml (U/ml). Concentration dependent responses could be observed as early as 6 h but greater sensitivity was obtained at 24 h. Neutralizing antibodies to IFNα and IFNß reduced the response to baseline. As proof of principle supernatants from RAW 264.7 (murine macrophage; parental cell line) infected with 1 multiplicity of infection (moi) of influenza A virus (X31/H3N2) were used as test samples. Pre-treatment of the supernatant with anti-IFNα failed to reduce the cell response but it was reduced to background by anti-IFNß. The high level of IFNß but very low level of IFNα was confirmed by ELISA. Availability, ease of use and maintenance, and possible cost savings make application of this reporter gene cell approach a valuable alternative to other methods for measuring Type I interferon.