Anti ifn γ antibody clone 1 d1k

PBMCs were pulsed either with 1 µg/ml or with 5 µg/ml of HLA class I or HLA class II peptide, respectively. Irrelevant peptides with the respective HLA restrictions were used as negative control (YLLPAIVHI for HLA-A*02 (source protein: DDX5_HUMAN), and ETVITVDTKAAGKGK for HLA class II (source protein: FLNA_HUMAN)). Cells were cultured for 12 days adding 20 U/ml IL-2 (Novartis, Basel, Switzerland) on days 2, 5, and 7. Peptide-stimulated PBMCs were analyzed by IFN-γ enzyme-linked immunospot (ELISPOT) assay on day 12^{67 (link)}, with anti-IFN-γ antibody (clone 1-D1K, 2 µg/mL, MabTech), anti-IFN-γ biotinylated detection antibody (clone 7 B6 1, 0.3 µg/mL, MabTech), ExtrAvidin-Alkaline Phosphatase (1:1000 dilution, Sigma-Aldrich) and BCIP/NBT (5 bromo 4-chloro 3 indolyl-phosphate/nitro-blue tetrazolium chloride, Sigma-Aldrich). Spots were counted using an ImmunoSpot S6 analyzer (CTL, Cleveland, OH, USA) and T cell responses were considered positive if >10 spots/500,000 cells were counted, and the mean spot count was at least three-fold higher than the mean spot count of the negative control.

Blood samples were collected before and every 3 cycles on treatments. PBMC were isolated and frozen for batched subsequent analysis. Direct ex vivo interferon (IFN)-γ Elispot analysis was performed using cryopreserved PBMC to assess for induction of antigen-specific CD8⁺ T cell responses. Briefly, one vial of frozen PBMCs from each time point was thawed and plated into anti-IFN-γ antibody (clone 1-D1K, Mabtech, Inc) pre-coated Elispot plate at 500,000 per well. Cells were then stimulated with either EBV control peptide (Invitrogen, Inc.) or one of the melanoma peptides, gp-100, Mage-3, Melan A, and NA-17 (Multiple Peptide Systems), at 50 μM for 24 h at 37 °C. Medium alone was used as negative control and P + I (PMA + Ionomycin) was used as positive control. For P + I control wells, 5000 PBMC were plated per well. After 24 h, plates were washed and incubated with a biotinylated anti-IFN-γ secondary Ab (clone 7-B6-1, Mabtech, Inc) for 2 h at room temperature, washed again and incubated with streptavidin-conjugated AP for 1 h, washed, and incubated with AP substrate. Excess substrate was removed by rinsing with tap water. Plates were then captured and counted using a CTL-ImmunoSpot S6 Core Analyzer from Cellular Technology Ltd (Cleveland, OH). All samples were analyzed in triplicate, and the mean response to the negative control was subtracted from each sample.

IFNγ ELISPOT was performed as previously described (5 (link), 6 (link)). Briefly, 15,000 TCR-transduced or mock-transduced (TEG-LM1) T cells and 50,000 target cells (ratio 0.3:1) were cocultured for 24 h in nitrocellulose-bottomed 96-well plates (Merck, Schiphol-Rijk, the Netherlands), pre-coated with anti-IFNγ antibody (clone 1-D1K) (Mabtech, Nacka Strand, Sweden). Plates were washed and incubated with a second biotinylated anti-IFNγ antibody (clone 7-B6-1) (Mabtech) followed by streptavidin-HRP (Mabtech). IFNγ spots were visualized with tetramethylbenzidine substrate (Sanquin) and the number of spots was quantified using ELISPOT Analysis Software (Aelvis, Hannover, Germany). IFNγ ELISA was performed using ELISA-ready-go! Kit (eBioscience) following manufacturer’s instructions. Effector and target cells (E:T 15,000:15,000) were incubated for 24 h in the presence of pamidronate when indicated.