Human ifn γ elispotpro kit

ELISpot assay was performed using Human IFN-γ ELISpot^PRO kit (Mabtech) according to the manufacturer’s instructions. Briefly, plates were washed four times with PBS and blocked with CellGenix GMP DC Medium containing 10% of ABS at least 30 min before use. For co-culture, 1 × 10^4 responder CD8⁺ T cells for detection of CD8⁺ T-cell responses were co-cultured with 5 × 10^3 stimulator autologous BMDCs. Responder CD8⁺ T cells were educated by iPSDCs-neoantigen peptide or iPSDCs-CTOS ivtRNA. Autologous PBMCs were generated using the plastic adherence method, as described previously^{34 (link)} and then pulsed with the respective neoantigen peptides (20 μg/ ml). After that, they were directly added to the ELISpot wells and incubated with responder CD8⁺ T cells overnight in DC Medium containing 10% of ABS at 37 °C. The mAb CD3-2 was used as a positive control. After 24 h of co-culture, cells were removed from the plate, washed five times with PBS, and then diluted with anti-human IFN-γ mAb (7-B6-1-Biotin, Mabtech) 1:200 in filtered PBS containing 0.5% ABS. After rinsing, TMB substrate solution was used to develop the immunospots. Spots were captured and analyzed by an automated ELISpot reader, ImmunoSpot S4 Software package, Version 6.0.0.2 (Cellular Technology Limited).

PepMix pools containing 15-mer peptides overlapping by 10 amino acids from either SARS-CoV-2 spike S1 or S2, N/M/E protein domains were purchased from Alta Biosciences (University of Birmingham). T cell responses of postvaccination samples to the above peptide mixes were determined using a Human IFN-γ ELISpot PRO kit (Mabtech). Isolated PBMCs were thawed and rested overnight before assay in R10 (RPMI 1640 + 10% FCS + penicillin/streptomycin). Then, 2–3 × 10⁵ PBMCs were stimulated in duplicate with peptide mixes at 1 μg ml⁻¹ per peptide, anti-CD3 and CEFX cell stimulation mix (JPT) as a positive control or DMSO as a negative control for 16–18 h. Supernatants were collected and stored at −80 °C. After the development of plates according to the manufacturer’s instructions, plates were read using the BIOSYS Bioreader 5000. Mean spot counts in DMSO-treated negative control wells were deducted from the means to generate normalized spot counts for all other treated wells. Cutoff values were determined previously^{14 (link)}.
Cytokine concentrations in the ELISpot supernatants were assayed using a LEGENDplex Human Th Panel (BioLegend) according to the manufacturer’s instructions. Data were analyzed using the LEGENDplex Data Analysis Software Suite (BioLegend).

The numbers of IFN-γ+ T lymphocytes induced by the PP19128R vaccine and our previously developed TB vaccine HP13138PB [49 (link)] were determined using an ELISpot assay. First, a 5 mL blood sample was collected from HCs (n = 21), LTBI individuals (n = 25), and ATB patients (n = 19). PBMCs were separated from the blood sample using a human lymphocyte separation medium (Solarbio, Beijing, China, Cat: P8610) according to the manufacturer’s instructions. Then, 2.5 × 10⁵ PBMCs in 100 μL GIBCO AIM-V medium (Life Technology Invitrogen, California, USA, Cat. No. 087-0112DK) were added to one well of a 96-well ELISpot culture plate and incubated with 50 μL PP19128R or HP13138PB vaccine (100 μg/mL) or 50 μL AIM-V medium (negative control) in a CO₂ incubator at 37 °C for 24 h. The number of spot-forming cells (SFCs) was detected using a Human IFN-γ ELISpot ^PRO kit (Mabtech AB, Nacka Strand, Sweden, Cat. No. 3420-2APW-10) according to the manufacturer’s instructions.