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Measuring type I interferon using reporter gene assays based on readily available cell lines
1Scientific Research Department, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.
Journal of Immunological Methods
|June 13, 2018
Summary
Reporter gene assays using engineered cell lines offer a sensitive and cost-effective method for quantifying Type I interferons (IFNα/β). These assays provide a valuable alternative to traditional methods like ELISAs for interferon detection.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Traditional methods for detecting Type I interferons (IFN), such as ELISAs, can be complex and costly.
- Reporter gene assays (RGAs) offer a promising alternative for quantifying biological responses.
- Commercial RGAs responsive to IFN exposure are becoming increasingly available.
Purpose of the Study:
- To evaluate the utility of RAW-Blue ISG™ and B16-Blue IFNα/β™ reporter cell lines for quantifying Type I interferons (IFNα and IFNβ).
- To compare the sensitivity and kinetics of these RGAs with existing interferon detection methods.
Main Methods:
- Utilized RAW-Blue ISG™ and B16-Blue IFNα/β™ cells, which produce secreted embryonic alkaline phosphatase (SEAP) in response to IFN.
- Quantified SEAP levels using spectrophotometry/luminometry after exposure to varying concentrations of IFNα and IFNβ.
- Assessed the response kinetics and effect of neutralizing antibodies against IFNα and IFNβ.
- Validated the assay by measuring IFN levels in supernatants from influenza A virus-infected macrophages.
Main Results:
- RAW-Blue ISG™ and B16-Blue IFNα/β™ cells exhibited a log-linear response to IFNα/β over a 4-log concentration range (10-100,000 U/ml).
- Concentration-dependent responses were detectable as early as 6 hours, with enhanced sensitivity at 24 hours.
- Neutralizing antibodies confirmed the specificity for IFNβ in influenza virus-infected samples, aligning with ELISA results.
- The reporter gene assay demonstrated a high level of IFNβ and low IFNα production in infected cell supernatants.
Conclusions:
- RAW-Blue ISG™ and B16-Blue IFNα/β™ reporter cell lines provide a sensitive, reproducible, and potentially cost-effective method for measuring Type I interferons.
- These RGAs represent a valuable alternative to conventional assays like ELISAs for interferon quantification.
- Ease of use, maintenance, and potential cost savings support the adoption of this reporter gene cell approach in research and diagnostics.
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