Computational protocol: Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

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Protocol publication

[…] A SEAP reporter gene assay based on expression plasmid containing an IFN-inducible promoter (pNiFty2) was used to assess the biological activity of glycosylated HEK293-produced IFNα2b in comparison to non-glycosylated IFNα2b. HEK293 cells were transfected with the pNiFty2 reporter plasmid, which encodes the secreted embryonic alkaline phosphatase (SEAP) under the control of the human ISG56 promoter. Transfected cells were plated in 96 well plates at a cell density of 105 cells/mL and stimulated, 24 h post-transfection, with IFNα2b at the indicated concentrations. Following an additional 48 h period of incubation, the supernatants were collected and assayed for SEAP activity. The hydrolysis of paranitrophenyl phosphate (pNPP) was measured as a function of time to determine SEAP activity induced with IFN treatments, as previously described []. The SEAP activity is expressed as the increase in absorbance units at 410 nm per minute.Antiviral assays were carried out by PBL InterferonSource (Piscataway, NJ) using Madin-Darby Bovine Kidney (MDBK) cells challenged by Vesicular stomatitis virus or human A549 cells challenged with encephalomyocarditis virus []. Cells were incubated with two-fold serial dilutions of IFNα2b standard (PBL InterferonSource), 293-IFNα2b, or control (Media). After 24 h incubation with the virus, cell viability was determined via crystal violet staining (570 nm absorbance). The sample titer (IC50), calculated by SigmaPlot software (SPSS Inc., Point Richmond, CA), was based on the 50% cytopathic effect of the assay. Units of anti-viral activity were based on the titer of a PBL lab standard, which was determined against the NIH reference standard for human IFNα2b (Gxa01-901-535). […]

Pipeline specifications

Software tools SigmaPlot, SPSS
Application Miscellaneous
Organisms Homo sapiens