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Updated: Sep 28, 2025
High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes
Published on: March 24, 2015
Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means
Michael Puckette1, J Barrera2, M Schwarz3
1Plum Island Animal Disease Center, U. S. Department of Homeland Security Science and Technology Directorate, P.O. Box 848, Greenport, NY, 11944, USA. michael.puckette@st.dhs.gov.
Insights
This study developed a rapid and robust assay for measuring interferon concentrations. The novel method uses luciferase activity for quick and accurate quantification, outperforming traditional ELISA assays.
Area of Science:
- Biotechnology
- Immunology
- Molecular Biology
Background:
- Type I interferons are crucial in research and therapeutics.
- Existing assays (plaque reduction, ELISA) are costly, complex, and slow.
- Need for a faster, more robust method to quantify interferon levels.
Purpose of the Study:
- Develop a rapid and robust assay for interferon concentration determination.
- Evaluate direct and indirect quantification methods using novel constructs.
- Improve upon limitations of current interferon measurement techniques.
Main Methods:
- Engineered bi-cistronic constructs for indirect quantification (luciferase-2A-interferon).
- Created fusion proteins for direct quantification (luciferase-interferon).
- Assessed luminescence, ELISA concentration, and antiviral activity in transfected cell cultures.
Main Results:
- Bi-cistronic constructs showed correlated luciferase and antiviral activity.
- Fusion proteins retained luminescence; only interferon α fusion had comparable antiviral activity.
- Strong linear correlation observed between luminescence and interferon concentration over a wide dynamic range.
Conclusions:
- The developed assay, using both direct and indirect methods, rapidly quantifies recombinant interferon concentrations.
- This approach offers a wider dynamic range compared to ELISA-based assays.
- The correlation of antiviral and luciferase activities validates the assay's utility.
Background:
Type I interferons are widely used in research applications and as biotherapeutics. Current assays used to measure interferon concentrations, such as plaque reduction assays and ELISA, are expensive, technically challenging, and may take days to provide results. We sought to develop a robust and rapid assay to determine interferon concentrations produced from transiently transfected cell cultures.
Method:
Indirect quantification of recombinant interferon was evaluated using a novel bi-cistronic construct encoding the Foot-and-mouth disease virus 2A translational interrupter sequence to yield equimolar expression of Gaussia princeps luciferase and porcine interferon α. Direct quantification was evaluated by expression of a novel fusion protein comprised of Gaussia princeps luciferase and porcine type I interferon. Plasmids encoding constructs are transiently transfected into cell cultures and supernatant harvested for testing of luminescence, ELISA determined concentration, and anti-viral activity against vesicular stomatitis virus.
Results:
Bi-cistronic constructs, utilized for indirect quantification, demonstrate both luciferase activity and anti-viral activity. Fusion proteins, utilized for direct quantification, retained secretion and luminescence however only the interferon α fusion protein had antiviral activity comparable to wildtype porcine interferon α. A strong linear correlation was observed between dilution and luminescence for all compounds over a dynamic range of concentrations.
Conclusion:
The correlation of antiviral and luciferase activities demonstrated the utility of this approach, both direct and indirect, to rapidly determine recombinant interferon concentrations. Concentration can be determined over a more dynamic concentration range than available ELISA based assays using this methodology.
