Plum Island Animal Disease Center, U. S. Department of Homeland Security Science and Technology Directorate, P.O. Box 848, Greenport, NY, 11944, USA.
Plum Island Animal Disease Center, Leidos, Inc., P.O. Box 848, Greenport, NY, 11944, USA.
BMC Biotechnol. 2022 Mar 29;22(1):13. doi: 10.1186/s12896-022-00743-9.
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.
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.
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.
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.
I 型干扰素广泛应用于研究应用和生物治疗。目前用于测量干扰素浓度的检测方法，如蚀斑减少检测和 ELISA，既昂贵又具有技术挑战性，并且可能需要数天才能提供结果。我们试图开发一种强大而快速的测定法来确定瞬时转染细胞培养物产生的干扰素浓度。
使用新型双顺反子构建体评估重组干扰素的间接定量，该构建体编码口蹄疫病毒 2A 翻译中断序列，以产生等量表达的 Gaussia 萤光素酶和猪干扰素 α。通过表达由 Gaussia 萤光素酶和猪 I 型干扰素组成的新型融合蛋白来评估直接定量。将编码构建体的质粒瞬时转染到细胞培养物中，并收获上清液进行发光检测、ELISA 确定浓度和抗水疱性口炎病毒活性检测。
用于间接定量的双顺反子构建体均显示出萤光酶活性和抗病毒活性。用于直接定量的融合蛋白保留了分泌和发光活性，但只有干扰素 α 融合蛋白具有与野生型猪干扰素 α 相当的抗病毒活性。在浓度的动态范围内，所有化合物的稀释度与发光度之间均观察到强线性相关性。
抗病毒和萤光酶活性的相关性表明了这种直接和间接方法快速确定重组干扰素浓度的实用性。与基于 ELISA 的现有检测方法相比，该方法可在更广泛的浓度范围内确定浓度。
