Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means
Abstract
Abstract 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 ofmore »
- Authors:
- Publication Date:
- Research Org.:
- Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN (United States)
- Sponsoring Org.:
- USDOE; US Department of Homeland Security (DHS)
- OSTI Identifier:
- 1859789
- Alternate Identifier(s):
- OSTI ID: 1905035
- Grant/Contract Number:
- AC05-06OR23100; HSHQDC14-F-00035; GS-23F-80006H
- Resource Type:
- Published Article
- Journal Name:
- BMC Biotechnology (Online)
- Additional Journal Information:
- Journal Name: BMC Biotechnology (Online) Journal Volume: 22 Journal Issue: 1; Journal ID: ISSN 1472-6750
- Publisher:
- Springer Science + Business Media
- Country of Publication:
- United Kingdom
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Gaussia luciferase; interferon α; interferon β; fusion protein; VSV; FMDV; luciferase; assay; anti-viral
Citation Formats
Puckette, Michael, Barrera, J., Schwarz, M., and Rasmussen, M. Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means. United Kingdom: N. p., 2022.
Web. doi:10.1186/s12896-022-00743-9.
Puckette, Michael, Barrera, J., Schwarz, M., & Rasmussen, M. Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means. United Kingdom. https://doi.org/10.1186/s12896-022-00743-9
Puckette, Michael, Barrera, J., Schwarz, M., and Rasmussen, M. Tue .
"Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means". United Kingdom. https://doi.org/10.1186/s12896-022-00743-9.
@article{osti_1859789,
title = {Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means},
author = {Puckette, Michael and Barrera, J. and Schwarz, M. and Rasmussen, M.},
abstractNote = {Abstract 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.},
doi = {10.1186/s12896-022-00743-9},
journal = {BMC Biotechnology (Online)},
number = 1,
volume = 22,
place = {United Kingdom},
year = {Tue Mar 29 00:00:00 EDT 2022},
month = {Tue Mar 29 00:00:00 EDT 2022}
}
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