Ifn γ secreting elispot assay

Antigen-specific T lymphocyte responses were detected via an IFN-γ-secreting ELISPOT assay (BD). Briefly, 96-well ELISPOT plate membranes were coated with anti-human IFN-γ antibody at 4°C overnight before use. PBMCs from donors were incubated in wells (2.5 × 10⁵/well ex vivo for freshly isolated PBMCs and 5 × 10⁴/well for in vitro-cultured T-cell lines) along with viruses (multiplicity of infection [MOI] of 3) or peptide pools (2 µM for individual peptides) for 18 h at 37°C with 5% CO₂, as well as with phytohemagglutinin (PHA) as a positive control for nonspecific stimulation. Cells incubated without stimulation were employed as a negative control. After incubation, cells were removed and, in turn, plates were processed with biotinylated IFN-γ detection antibodies, streptavidin-horseradish peroxidase (HRP) conjugate, and substrate. The development of the spots was stopped by thoroughly rinsing with water. The numbers of the spots were captured and quantified with an automatic ELISPOT reader and image analysis software (Cellular Technology Limited). The threshold of the positive responses set for ex vivo ELISPOT was ≥20 SFCs/10⁶ PBMCs.

ELISPOT assay was performed using an IFN-γ secreting ELISPOT assay (BD Bioscience, San Jose, California) as previously described (53 (link)). In brief, 96-well ELISPOT plate was pre-incubated with anti-IFN-γ coating antibody overnight at 4°C, and then blocked for 2 h at RT. With TCR-transduced T cells as effector cells and SCT-K562 cells as target cells, the effector cells and target cells were co-incubated in pre-incubated plates with different ratios (10:1; 5:1; 1:1; 1:5; and 1:10) with a total of 2 × 10⁴ cells per well. Effector cells incubated with PMA/ION were added as positive controls, and effector cells incubated with medium were employed as negative controls. After incubation of 18 h, cells were removed, and the plates were processed according to the instructions of the manufacturer (BD Life Sciences, San Jose, California). The number of spots were determined using an automatic ELISPOT reader and image analysis software (Cellular Technology Ltd., Ohio).