Anti human ifn γ mab 7 b6 1

T cells were incubated with autologous DCs alone (control) or with DCs incubated with rFVIII (0.2 µM), ovalbumin (3 µM), and KLH (1 µM) or PBMCs for FVIII peptides (10 µg/ml) in MultiScreen 96-well plates (Merck Millipore) previously coated with 2.5 µg/mL of anti-human IFN-γ monoclonal antibody (mAb; 1-D1K; Mabtech). The T cell lines and APCs were plated at equal number for both stimulated and unstimulated control conditions. After overnight incubation, spots were revealed using 0.25 µg/mL of biotinylated anti-human IFN-γ mAb (7-B6-1; Mabtech) in phosphate-buffered saline/bovine serum albumin 1%, extravidin-phosphatase (dilution 1:3,000 in phosphate-buffered saline/Tween20 0.05%/bovine serum albumin 1%; Sigma-Aldrich), and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate-toluidine salt (Sigma-Aldrich). The spot number was determined by using the ELISPOT Reader System (Autoimmun Diagnostika, Ebinger, Germany). HLA restriction was assessed using monoclonal antibodies (10 μg/ml) specific to HLA-DP (B7/21), HLA-DQ (SPVL3), or HLA-DR (L243).

Fifty thousand cells from the short-term culture or a mix of 50,000 cells from T-cell lines and 50,000 autologous PBMCs were incubated with HA or analogs (10 μg/mL) in Multiscreen 96-well plates (Merck Millipore, Bedford, MA, USA) previously coated with 2.5 μg/mL anti-human IFN-γ monoclonal antibody (mAb, 1-D1K; Mabtech, Nacka, Sweden). After overnight incubation, spots were revealed using 0.25 μg/mL biotinylated anti-human IFN-γ mAb (7-B6-1; Mabtech) in phosphate-buffered saline/1% bovine serum albumin, extravidin-phosphatase (dilution 1:3,000 in phosphate-buffered saline/0.05% Tween 20/1% bovine serum albumin; Sigma-Aldrich, Saint-Quentin Fallavier, France), and NBT/BCIP (Sigma-Aldrich). Spot number was determined by the AID ELISPOT Reader System (AID GmbH, Ebinger, Germany). PHA was introduced in the assay as positive control. T-cell responses was considered as specific when a spot count was 2-fold higher in the presence of the peptide than in its absence, with a minimal difference of 25 spots.