Anti mouse ifn γ antibody an18

The CTL responses were evaluated by γ‐interferon (IFN‐γ) enzyme‐linked immunospot (ELISPOT) assay. Five mice in each treatment group were immunized once per week for 2 weeks. Spleen cells were collected from the vaccinated B6 mice 1 week after the last vaccination and resuspended in red blood cell lysis buffer, then resuspended at 10⁷ cells/mL in X‐VIVO15 (Lonza, Walkersville, MD, USA) supplemented with 10 mM HEPES, 2 mM L‐alanyl‐L‐glutamine (nacalai tesque, Kyoto, Japan), and 0.05 mM 2‐mercaptoethanol. Spleen cells (10⁶ in 0.1 mL) were seeded in each well of a 96‐well Multi‐Screen Filter Plate (MSHAS4510; Merck Millipore, Darmstadt, Germany) coated by anti‐mouse IFN‐γ antibody (AN18; Mabtech, Nacka Strand, Sweden). An equal volume of X‐VIVO 15 with or without 20 μg/mL OVA_257–264 was added to each well. After 18 h of incubation at 37°C, the plate was incubated with biotin‐conjugated anti‐mouse IFN‐γ (R4‐6A2; Mabtech) at room temperature for 2 h, followed by 1 h of incubation with ExtrAvidin alkaline phosphatase (Sigma‐Aldrich, St. Louis, MO, USA) at room temperature. γ‐Interferon spots were visualized by SIGMAFAST BCIP/NBT (Sigma‐Aldrich). The ELISPOT plates were analyzed by ImmunoCapture version 6 (Cellular Technology, Shaker Heights, OH, USA).

ELISPOT assays were performed according to the manufacturer’s protocol (Mabtech, Nacka Strand, Sweden) to detect IFN-γ-producing T cells in the splenocytes. Briefly, 96-well ELISPOT plates (Millipore, Burlington, MA, USA) were pre-coated with anti-mouse IFN-γ antibody (AN18, Mabtech) at 4°C overnight. After removing the coating antibody, wells were blocked with RPMI 1640 medium containing 10% FBS for 1 h. The splenocytes isolated from immunized mice were added to ELISPOT plates and then stimulated with four peptide pools spanning DENV-2 E80 protein (P1, P2, P3, and P4), a single immunodominant peptide (P_265–273) from DENV-2 NS1, or PBS as negative control at 37°C for 48 h. After incubation, the wells were washed five times with PBS and then incubated with a biotinylated anti-mouse IFN-γ detection antibody (R4-6A2-biotin; Mabtech) diluted in PBS containing 0.5% FBS at 0.2 μg per well for 2 h at room temperature, followed by the addition of diluted alkaline-phosphatase-conjugated streptavidin for 1 h. Immune spots were developed using TMB substrate and counted with the ImmunoSpot Analyzer (CellularTechnology, Kennesaw, GA, USA).