Method for quantification of porcine type I interferon activity using luminescence, by direct and indirect means

Figures

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  Fig. 1. Schematic diagram of methodology utilizing luminescence to quantify IFNα. A Indirect quantification is achieved through the FMDV 2A translational interrupter sequence, blue, that results in expression of luciferase and IFNα as separate proteins in an equimolar ratio in cell culture media. B Direct quantification is achieved utilizing a fusion protein consisting of SGLuc and IFNα that is secreted into cell culture media. The SGLuc-IFNα fusion protein retains both luminescence and anti-viral activity

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  Fig. 2. A Diagrams of bi-cistronic SGLuc-Δ1D2A-IFNα and IFNα-Δ1D2A-SGLucΔ1M constructs evaluated for correlation of luciferase and antiviral activities. Pink box represents the IFNα secretion peptide sequence. B Western blots using anti-porcine IFNα and anti-GLuc antibodies demonstrated protein banding patterns with expected sizes and confirmed secretion into cell culture media. Blots were cropped to focus on results and representative of N = 2 replicates; uncropped full-length blots are included in Additional file 1: Fig. S1. C Luminescence of serially diluted media, N = 5, samples from cultures producing SGLuc-Δ1D2A-IFNα and IFNα-Δ1D2A-SGLuc Δ1M. Sample dilutions range from 1/50 to 1/6400. Dotted lines represent the line of best fit

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  Fig. 3. A Diagrams of mono-cistronic constructs expressing SGLuc-IFNα, SGLuc-IFNβ, IFNα, and IFNβ. In SGLuc-IFN constructs, the native secretion domains of porcine IFNα and IFNβ, pale red and pale green respectively, are replaced with the amino acid sequence for SGLuc. B Western blots of media from transfected cell cultures expressing native porcine IFN and chimeric SGLuc-IFNs using anti-IFNα, anti-IFNβ, and anti-GLuc antibodies demonstrate expression and secretion into cell culture media. Blots are cropped to focus on results and representative of N = 3 replicates; uncropped full-length blots are included in Additional file 1: Fig. S2

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  Fig. 4. A Luciferase readings for serial dilutions of SGLuc-IFNα and β samples show strong linear correlation over tested dilutions of 1/50 to 1/6400. B Luciferase activity, N = 7 replicates per sample, in media produced from three separate transfections of SGLuc-IFNα expressing plasmid. Corresponding mean plaque numbers for each sample are within bars with standard deviation in parenthesis below; N = 3 replicates per sample. RLUs/0.5 s, relative luciferase units per half second. C Triplicate wells for plaque assays evaluating SGLuc-IFNα efficacy against FMDV. Tested dilutions demonstrate a reduction in plaque numbers from untreated control values of 200 to an average of 14.7 and 29.3 for dilutions of 1:1600 and 1:3200, respectively