Anti mouse ifn γ capture antibody

Spleen cells were isolated from the immunized mice and suspended at a final concentration of 5 × 10⁶ per mL in RPMI 1640 medium (Gibco) supplemented with 10% (vol/vol) heat-inactivated FBS (Gibco), antibiotic–antimycotic solution (anti-anti; Gibco), 2 mM Glutamax (Gibco), 1 mM sodium pyruvate (Gibco), and 55 μM 2-mercaptoethanol (Gibco). Multiscreen-HA filter plates were coated with anti-mouse IFN- γ capture antibody (R&D systems) at 4 °C overnight and blocked with RPMI 1640 medium with 10% FBS. Cells were added with intact virions (VZV lysate; Mycrobix), recombinant glycoprotein E (gE), glycoprotein I (gI) (gE and gI from Peptron), IE63 (Genscript), and overlapped peptide (IE63 OLP from JPT), and incubated overnight at 37 °C. The plates were washed with PBS (3x) and biotinylated anti-mouse IFN- γ detection antibody (R&D systems) was added. After washing, streptavidin was conjugated and 3-amino-9-ethylcarbazole (AEC; BD) were added to the plates. The reaction was stopped by rinsing with tap water. Spot-forming units (SFU) were read using ELISPOT reader (Autoimmune Diagnnostika). Adjusted SFU were obtained by subtraction of mock-stimulated counts (mock lysate for VZV lysates, medium for recombinant protein, and DMSO for OLP).

MultiScreen filter 96-well-plates (Millipore) were coated with the anti-mouse IL4 or anti-mouse IFNγ capture antibody (R&D Systems) and incubated overnight at 4°C. The plates were then blocked with 200 μL of RPMI-10 medium (RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μM 2-mercaptoethanol, and 2 mM L-glutamine) and incubated for 2 h at 37°C. In parallel, spleens were harvested from mice at day 5 post-A2 challenge and prepared to a single cell suspension. The cell suspensions were collected by centrifugation for 10 min at 200 × g and suspended in RPMI-10 at 10⁷ cells/mL. Spleen cell suspensions were added to the wells, and cells were stimulated with either 10 μg/mL RSV M2 _(82−90) peptide, 10 μg/mL RSV F _(51−66) peptide, 10 μg/mL RSV G _(183−198) peptide, or 10 μg/mL eGFP _(200−208) (irrelevant peptide control) for 24 h at 37°C and 5% CO₂. Plates were washed 4 times with wash buffer (0.05% Tween-20 in PBS), anti-mouse IL4 or anti-mouse IFNγ detection antibody (R&D Systems) was added, and plates were incubated overnight at 4°C. Detection antibody was removed, plates were washed, and cytokine spots were developed using NBT/BCIP substrate (R&D Systems). Spots were enumerated using an ELISPOT reader (AID, San Diego).