Human ifn γ elisa kit

Tuberculosis-PE were incubated with 10 µg/ml of the following neutralizing antibodies for 1 h at 4°C for the specific depletion of IL-10 (αIL-10, clone 19F1; Biolegend), IL-6 (αIL-6, clone MQ2-13A5; BD Bioscience), IL-1β (αIL-1β, clone 8516.311; SIGMA), or TNF-α (αTNF-α, clone MAb1; Biolegend). Then, 100 µl/ml of Protein G Sepharose beads (Amersham) were added and incubated for 1 h at 4°C. Finally, TB-PE were centrifuged at 12,000 g to remove antibody-bead complexes and then filtered (0.22-µm pores) before use. IFN-γ depletion was performed by incubating TB-PE for 2 h in sterile 96-well plates that had been coated with the capture antibody provided by the Human IFN-γ ELISA Kit (BD Bioscience). In all cases, depletions were controlled by ELISA.

Levels of IFN-γ or TNF-α production were evaluated to determine whether tumor-suppressed T cells were reactivated. Human CD4+ T lymphocytes were co-cultured with A375 cells, MDA-MB-231 cells, PD-L1-negative HEK293T cells, or HEK293T cells with hPD-L1 expression (HEK293T-hPD-L1) in 96-well plates. A density of 10,000 cells per well was plated and adhered for 24 h; then, 20,000 reactivated CD4+ T lymphocytes per well were added to the plates with different inhibitors, followed by co-culturing at 37°C, 5% CO₂ for 48 h. The level of IFN-γ or TNF-α production in culture supernatants was detected by Human IFN-γ ELISA kit (BD Biosciences) and Human TNF-α ELISA kit (BD Biosciences) (for the expression and purification of rU1 snRNPA, binding ELISA, and competitive ELISA, in vitro anti-cancer assay, in vivo anti-melanoma assay, see the supplementary materials for details).

1 × 10⁵ T2 cells loaded with 10 μM cognate peptide or alanine (or serine) peptide variants were co-cultured for 18 h with 1 × 10⁵ bulk-transduced T cells in round-bottom 96-well plates containing 250 μl of culture medium per well. All conditions were assayed in duplicate. Supernatants were tested for secreted IFNγ using a human IFNγ ELISA Kit (BD Biosciences) and absorbance was read at 450 nm as described above. NLV peptide variants were ALVPMVATV, NANPMVATV, NLAPMVATV, NLVAMVATV, NLVPAVATV, NLVPMAATV, NLVPMVAAV, NLVPMVATA, and NLVPMVSTV. HA1 peptide variants were ALHDDLLEA, VAHDDLLEA, VLADDLLEA, VLHADLLEA, VLHDALLEA, VLHDDALEA, VLHDDLAEA, VLHDDLLAA, and VLHDDLLES. HA2 peptide variants were AIGEVLVSV, YAGEVLVSV, YIAEVLVSV, YIGAVLVSV, YIGEALVSV, YIGEVAVSV, YIGEVLASV, YIGEVLVAV, and YIGEVLVSA. Variant peptides were synthesized by Severn Biotech Ltd.

PBMC were washed in pre-warmed RPMI 1640 supplemented with 2 mM L-glutamine, 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (complete RPMI) (all from Corning cellgro, Manassas, VA, USA), seeded in a 48-well cell culture plate at a density of 1 × 10⁴ cells/well in complete RPMI, and stimulated with 1μg/ml of the SARS-CoV-2 MP_S peptide pool or 0.1% DMSO (vehicle control). Cell culture plates were incubated for 24 hrs at 37°C in a 5% CO₂ humidified atmosphere. Each sample was tested in duplicate. As positive control, two wells per sample were treated with a mixture of 25 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) and 0.5 μM ionomycin calcium salt (Enzo Life Sciences, Farmingdale, CT, USA) for 2 hrs. Supernatants were collected and levels of IFNγ in supernatants were assayed using a commercial Human IFNγ ELISA kit (BD Biosciences, San Jose, CA), according to manufacturer’s instructions.