Anti human ifn γ 1 d1k

White-bottom ELISpot HTS plates (MSIPS4W10, Millipore) were hydrophilized with 35% EtOH and subsequently coated with anti-human IFN-γ (1-D1K, Mabtech). Blocking was performed with X-Vivo-20 (Lonza) supplemented with 1% human albumin (Sigma-Aldrich). PBMCs were thawed and rested overnight in X-Vivo-20. Per well, 4 × 10⁵ cells were seeded and stimulated with 2 μg of peptide in a total volume of 100 μl. H3K27M (p14–40), H3wt (p14–40), or MOG (p35–55) was used as a stimulant, and 10% DMSO diluted in aqua ad iniectabilia (Braun) at equal volume was added as negative control. Positive controls were 1-μg staphylococcal enterotoxin B (Sigma-Aldrich), 0.05-μg CMV with 0.05-μg AdV per well, and CEFX Ultra SuperStim Pools of mixed, known peptide epitopes for MHC I– and MHC II–specific stimulation (PM-CEFX-4 and PM-CEFX-3, respectively; JPT Peptide Technologies). After 44 hours, detection of IFN-γ–secreting cells was performed by adding biotinylated anti-human IFN-γ antibodies (7-B6–1), streptavidin-ALP (both Mabtech), and ALP color development buffer (Bio-Rad). IFN-γ Spots were quantified by an ImmunoSpot Analyzer (ImmunoSpot/CTL Europe).

ELISpot white-bottom multiscreen HTS plates (MSIPS4W10, Millipore) were coated with anti-human IFNγ (1-D1K, Mabtech) and blocked with X-Vivo-20 (Lonza) containing 2% human albumin (HA). PBMCs were thawed, rested overnight in X-Vivo medium and seeded at 4 × 10⁵ cells per well and stimulated with 2 μg peptides per well in 100 μl volume. PBMCs were stimulated with IDH1(R132H) (p123–142), wild-type IDH1 (p123–142), or MOG (p35–55) at equal concentrations or with peptide diluent aqua ad iniectabilia (Braun) with 10% DMSO (vehicle) at equal volume as negative controls, or with 1 μg staphylococcal enterotoxin B (Sigma-Aldrich) per well and 0.05 μg CMV with 0.05 μg AdV per well (both in 100 μl volume) as positive controls. After 40 h, IFNγ-producing cells were detected with biotinylated anti-human IFNγ antibodies (7-B6-1), streptavidin-ALP (both Mabtech) and ALP colour development buffer (Bio-Rad) and quantified using an ImmunoSpot Analyzer (Cellular Technology Ltd). Quality control was performed and reviewed by a second person. For categorization of T cell responses, transient T cell responses were defined as a spot count above 50 followed by a spot count of less than 50 at EOS. Sustained T cell responses were defined as a spot count above 50 followed by a spot count of more than 50 at EOS.