Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means
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 deve...
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| Format: | Article |
| Language: | English |
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BMC
2022-03-01
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| Series: | BMC Biotechnology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12896-022-00743-9 |
| _version_ | 1818354517512552448 |
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| author | Michael Puckette J. Barrera M. Schwarz M. Rasmussen |
| author_facet | Michael Puckette J. Barrera M. Schwarz M. Rasmussen |
| author_sort | Michael Puckette |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-13T19:26:41Z |
| format | Article |
| id | doaj.art-a09e8976bed246c6b59cd9580d548ac6 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-13T19:26:41Z |
| publishDate | 2022-03-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-a09e8976bed246c6b59cd9580d548ac62022-12-21T23:34:02ZengBMCBMC Biotechnology1472-67502022-03-012211810.1186/s12896-022-00743-9Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect meansMichael Puckette0J. Barrera1M. Schwarz2M. Rasmussen3Plum Island Animal Disease Center, U. S. Department of Homeland Security Science and Technology DirectoratePlum Island Animal Disease Center, Leidos, Inc.Oak Ridge Institute for Science and Education, Plum Island Animal Disease Center Research Participation ProgramPlum Island Animal Disease Center, U. S. Department of Homeland Security Science and Technology DirectorateAbstract 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.https://doi.org/10.1186/s12896-022-00743-9Gaussia luciferaseInterferon αInterferon βFusion proteinVSVFMDV |
| spellingShingle | Michael Puckette J. Barrera M. Schwarz M. Rasmussen Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means BMC Biotechnology Gaussia luciferase Interferon α Interferon β Fusion protein VSV FMDV |
| title | Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means |
| title_full | Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means |
| title_fullStr | Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means |
| title_full_unstemmed | Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means |
| title_short | Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means |
| title_sort | method for quantification of porcine type i interferon activity using luminescence by direct and indirect means |
| topic | Gaussia luciferase Interferon α Interferon β Fusion protein VSV FMDV |
| url | https://doi.org/10.1186/s12896-022-00743-9 |
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