Ifn γ elisa kit

PBMC (2 × 10⁵ per well) were stimulated for 24 h in U-bottom 96 well plates with BEI-BTV-8 (equivalent to 1 × 10⁵ pfu prior to inactivation), concanavalin-A (2.5 µg/mL) as the positive control, VP7 peptide (10 µg/mL), or an equivalent volume of DMSO as the negative control. IFN-γ production in culture supernatants was then tested using a commercially available IFN-γ ELISA kit (Mabtech, Sweden). The ELISA detection limit was 20 pg/mL. Data were normalized to 1 × 10⁶ PBMC.

IFN-γ secretion of T cell clones was measured in the supernatant using the IFN-γ ELISA kit (3420-1H-20, MABTECH) following the manufacturer’s protocol. Total IgM from mouse-serum was detected using the IgM-ELISA kit (3880-1AD-6, MABTECH) according to the manufacturer’s protocol. Serum samples were pre-diluted 1:10 000. IL-27 in cell culture supernatant was analyzed using the IL-27 (3458-1H-6, MABTECH) kit, following the manufacturers protocol. CXCl9, CXCL10, IL-6, and IL-10 were analyzed with a Magnetic Luminex assay kit (Bio-Techne, Minneapolis, MN, USA). The measurements were performed on a Bio-Rad BioPlex 200 system (Bio-Rad Laboratories, Basel, Switzerland) and data were analyzed with the Bio-Plex software 6.0 (version 6.0, Bio-Rad).

Twenty-four hours after TCR mRNA transfection, the T cells were stained with antibodies for CD3, CD8 and TCRVβ (BD Biosciences, San Jose, CA) of the respective TCR and analyzed by a FACSVerse flow cytometer (BD Biosciences, San Jose, CA). Dead cells were excluded by LIVE/DEAD Aqua dye (Life Technologies, Carlsbad, CA). The data was analyzed with the FlowJo V10.0.7 software (Tree Star, Ashland, OR). The NS3 H4 and F8 TCRs, and the NS5A 69 and 19 TCRs were analyzed. Redirected T cells were thereafter co-cultured with gt1a NS3_1073-1081 peptide- or gt1b NS5A_1992-2000 peptide-loaded T2 cells overnight. IFN-γ secretion into supernatants was quantified using the IFN-γ ELISA kit (Mabtech, Nacka Strand, Sweden).
Antiviral efficacy in the respective anti-HCV TCRs were measured as previously described30 (link) in co-culture assays with Huh7^A2HCV^Rep cells that contains a stably replicating HCV reporter replicon. The replicon-driven luciferase activity was measured using the ONE-Glo Luciferase Assay System (Promega, Madison, WI). Data is presented as percentage of relative light unit (RLU) reduction compared to untreated Huh7^A2HCV^Rep cells alone (without co-culture). RLU values of non-luciferase cells (transfected T cells alone) were subtracted.