Multiscreen 96 well filter plate

The frequency of IFN-γ-producing T cells in tissue samples was assessed by ELISpot. MultiScreen^® 96-well filter plates (Merck; Rahway, NJ, USA) were wetted with 20 µL of 35% ethanol and washed with PBS. Anti-IFN-γ monoclonal antibody (mAb; clone AN18), 15 µg/mL in PBS, was added to each well and incubated for 16 h at 4 °C. Wells were blocked with cRPMI (2 h, 37 °C) and washed with PBS. 0.5 × 10⁵ lung cells or 1 × 10⁵ spleen cells were added to each well in the presence of 10 µg/mL antigen (see specific experiments) or 5 µg/mL concanavalin A (Sigma; Melbourne, Australia) in cRPMI. Plates were incubated at 37 °C, 5% CO₂ overnight. Wells were washed with PBS containing 0.01% Tween-20. Anti-IFN-γ-biotin mAb (clone XMG-1.2), 2.5 µg/mL in PBS, was added and incubated for 2 h at 37 °C. Then, the wells were washed as before and 1:1000 avidin–alkaline phosphatase (Sigma) in PBS added to each well and incubated for 45 min at room temperature, followed by washing as before. Next, 100 µL of AP substrate solution (Bio-Rad, Sydney, Australia) diluted in NPP buffer was added to each well and the plate was incubated until spots were visible, then washed with water. Spots were counted using an AID ELISpot reader (Melbourne, Australia) with the software ELISpot Reader version 6.0.

IFN-γ ELISPO assays were performed as previously described (1) Briefly, MultiScreen 96-well filter plates (Merck Millipore, Darmstadt, Germany) were coated with antihuman IFN-γ monoclonal antibody (U-Cytech, Netherlands, No. CT640-10) overnight at 4° C. The wells were washed with PBS and blocked with R10 medium (RPMI-1640, 0.05 mM, 2-mercaptoethanol, 1 mM sodium pyruvate, 2mM l-glutamate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 10% fetal bovine serum [FBS], and penicillin/streptomycin (1×)) for 2 h at 37° C. Freshly isolated PBMCs were plated and stimulated in the presence of SARS-COV-2 S1 or S2 or N peptide pools (GenScript, China) for 24 h at 37° C. SARS-COV-2 peptides are 20 amino acids in length, with ten amino acids overlaps between sequential peptides. The plates were washed with PBS-Tween 20 (PBST), followed by incubation with biotinylated antihuman IFN-γ detection antibody (U-Cytech, Netherlands, No. CT640-10) and alkaline phosphatase (AKP)-conjugated streptavidin (BD, USA, No. 554065). Spots were developed by incubating in NBT/BCIP (Pierce, USA) for 10 min. Spots were counted using an ELISPOT reader (Bioreader 4000, BIOSYS, Germany). The number of spot-forming cells (SFC) is reported as the number of antigen-specific IFN-γ-secreting cells per million PBMCs.