Human ifnγ elispot plus

Peripheral mononuclear cells (PBMCs) were stimulated in vitro with 15-mer peptides (overlapping by 11 amino acids) spanning the full-length Spike protein sequence of the indicated variants. Variant peptide pools (JPT Pepmix^TM) included the following changes to match published deletions/mutation in each variant: B.1.1.7 variant (delta69-70, delta144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H), B.1.351 variant (L18F, D80A, D215G, delta242-244, R246I, K417N, E484K, N501Y, D614G, A701V); P.1 variant L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and V1176F). Cells were incubated overnight with peptide pools at a concentration of 1 μg per ml per peptide in a precoated ELISpot plate, (MabTech, Human IFNγ ELISpot Plus). Cells were then washed off, and the plates were developed via a biotinylated anti-IFN-γ detection antibody followed by a streptavidin-enzyme conjugate resulting in visible spots. After plates were developed, spots were scanned and quantified using the CTL S6 Micro Analyzer (CTL) with ImmunoCapture and ImmunoSpot software. Values are shown as the background-subtracted average of measured triplicates. The ELISpot assay qualification determined that 12 spot forming units were the lower limit of detection. Thus, anything above this cutoff signal is an antigen-specific cellular response.

HBV-specific CTLs were detected using commercial enzyme-linked immunosorbent spot (ELISPOT) assay kits (Human IFN-γ ELISpot PLUS; MABTECH, Stockholm, Sweden). Briefly, 2 × 10⁵ peripheral blood mononuclear cells (PBMCs) from non-responders were harvested in 96-well plates containing Roswell Park Memorial Institute 1640 medium supplemented with 10% (v/v) FCS, 100 U/mL penicillin G, and 100 µg/mL streptomycin. The cells were further stimulated with either HBsAg (genotype C) or HBcAg (Beacle, Inc.) or anti-CD3 monoclonal antibodies as the positive control. After 72 h of incubation at 37 °C under 5% CO₂, 0.1 µg/well of the detection antibody was added to the samples; then, the plates were incubated for another 2 h at 22 °C. Subsequently, streptavidin horseradish peroxidase was added to the samples and incubated at 22 °C for 1 h. Finally, 100 μL/well of 3,3′,5,5′-Tetramethylbenzidine substrate solution was added to the samples, and the plates were incubated for another 5–10 min at 22 °C. The plates were washed with PBS, and spots were analyzed using the AID ELISPOT Reader 08 classic (Advanced Imaging Devices GmbH, Straßberg, Germany).