Elispot assay kit

D-galacto-D-mannan-mediated interferon γ (IFNγ) secretion with or without antigen (O PA2 or A YC) was assayed using commercial enzyme-linked immune absorbent spot (ELISpot) assay kits (R&D Systems, MN, USA) as per the manufacturer’s recommendations. Murine PECs or porcine PBMCs (5 × 10⁵ cells/well) were cultured in 96-well polyvinylidene fluoride-backed microplates containing a monoclonal capture antibody specific for murine or porcine IFNγ and stimulated with 2 μg/mL (final concentration) of antigen mixed with 0.625, 1.25, 2.5, and 5 μg/mL of D-galacto-D-mannan sequentially, at 37°C and 5% CO₂ for 18 h. Antigen and PBS were used as positive (PC) and negative controls (NC), respectively. Data were obtained using the ImmunoSpot ELISpot reader (AID iSpot Reader System; Autoimmune Diagnostika GmbH, Strassberg, Germany). The results were presented as spot-forming cells (SFC) per number of cells added to the well (7 (link), 29 (link)).

Splenocytes (n = 5 per group) were isolated from the vaccinated chickens on days 13 and 28 after the initial vaccination. HA-specific cells secreting Interferon gamma (IFN-γ) and Interleukin-4 (IL-4) were analyzed using commercial ELISpot assay kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, splenocytes (1.0 × 10⁶ cells/well) isolated from the vaccinated chickens were cultured in 96-well plates containing chicken IFN-γ or IL-4 and stimulated with 10 µg/mL of HA–specific peptide (ISVGTSTLNQRLVP) for 36 h in a humidified incubator at 37 °C with 5% CO₂. The plates were washed with sterile PBS and incubated with biotinylated goat anti-chicken IFN-γ or IL-4 antibodies overnight at 4 °C, followed by AP-conjugated streptavidin at room temperature for 2 h. The plates were washed, developed with BCIP/NBT, and counted using an ImmunoSpot ELISpot reader (Bio-Tek instrument Inc., Winooski, USA).

Mouse IFN-γ secretion in response to the stimulation of pullulanase peptides was determined using an ELISpot assay kit (R&D Systems) following the manufacturer’s protocols. Briefly, the final pullulanase peptide pool (Supplemental Methods) was diluted in the culture medium to a final concentration of 4 μg/mL. The diluted peptide pool was added to a 96-well plate (100 μL/well), and then 100 μL splenocytes (1 × 10⁷ cells/mL) was added to each well. Medium only and concanavalin A (Thermo Fisher Scientific) were used as negative and positive controls, respectively. Cells were incubated overnight at 37°C. The number of SFU in each well was counted using an automated ELISpot reader (ImmunoSpot Analyzer S6 Universal M2, CTL).

About 4x10⁶ THP-1 cells were seeded in T75 flasks and differentiated towards immature dendritic cells (iDC) over a 5-day culture in 20 mL serum-supplemented RPMI 1640 in the presence of 100 ng/mL hIL4 (R&D Systems, 204-IL-020/CF) and 100 ng/mL hGM-CSF (Sigma-Aldrich, #GF304). To allow full maturation towards mature dendritic cells (mDC), iDC were collected via centrifugation, resuspended in serum free RPMI 1640 media supplemented with 200ng/mL hIL4, 100ng/mL hGM-CSF, 20ng/mL hTNFα (Sigma-Aldrich, #GF314), and 200ng/mL ionomycin (Tocris Bioscience, 2092/1), and plated at density 10,000/well in the 96-well plates provided with the ELISPOT assay kit R&D Systems, #EL485, Minneapolis, MN. Cells were kept in culture for 1 day to allow differentiation towards mDC, before beginning the transfection with MessengerMax-formulated mRNA to induce expression of the E6-E7 fusion protein in the vaccine. The day following the transfection, human antigen specific E7_11-20 T cells (Charles River Laboratories, #ASTC-1099) were added to the 96-well plate at density 20,000 cells/well. ASTC and mDC cells were kept in coculture for an overnight. The plates were processed as per manufacturer (R&D Systems, #EL485)’s instructions and read under the CTL Immunospot Analyzer (ImmunoSpot^®). CD209 monoclonal antibody eB-h209 (eBioscience™) was used to characterize DC differentiation via Flow Cytometry.

Isoprinosine with or without antigen-induced IFNγ secretion was analyzed using a commercial ELISpot assay kit (R&D Systems, Minneapolis, MN, USA) as per manufacturer’s instructions. Murine PECs (5 × 10⁵ cells/well) or porcine PBMCs (5 × 10⁵ cells/well) were stimulated with 2 μg/mL (final concentration) of inactivated FMDV O PA2 or A YC antigen mixed with 0, 0.625, 1.25, 2.5, and 5 μg/mL of isoprinosine sequentially for 18 h in a humidified incubator at 37°C with 5% CO₂. PBS and antigen (O PA2 and A YC) were used as negative control (NC) and positive control (PC), respectively. Data were obtained using the ImmunoSpot ELISpot reader (Autoimmune Diagnostika GmbH, Strassberg, Germany).