Biotinylated anti ifnγ antibody clone 7 b6 1

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.

Anti-AAV2 and anti-AAV8 cellular immune responses were evaluated with an IFNγ ELISpot assay using overlapping peptide libraries covering the whole AAV2 or AAV8 capsid sequences (15 per 10 mers, PEPscreen, Sigma) split into three peptide pools each. MultiScreenHTS filter plates, with polyvinyldiene difluoride membrane (PVDF, Millipore) were coated overnight at 4°C with human anti-IFNγ antibody (clone MT126L, Mabtech). After coating, 2 × 10⁵ cells per well were plated and restimulated 48H with AAV2- or AAV8- derived peptide pools at a final concentration of 10 μg/mL. Medium alone served as negative control, while cells stimulated with concanavalin A (Con A, 10 μg/mL, SIGMA) served as a positive control. After incubation with a biotinylated anti-IFNγ antibody (clone 7-B6-1, MabTech) and Streptavidin-ALP (MabTech), enzymatic reaction was revealed using NBT/BCIP (MabTech). Spot number was determined using an ELISpot iSpot Spectrum reader (AID, Strassberg, Germany) and analyzed with AID ELISpot reader Software V7.0 (AID, Germany). Responses were considered positive when the number of spot-forming colonies per million cells was >50 and at least threefold higher than the medium alone negative control. For positive responses, statistical analyses were performed using a DFR(2×) test (Distribution Free Resampling).

IFN-γ-ELISpot assays were performed as described before (2 (link)). In short, approximately 100,000 pre-cultured cells were distributed into each well of 96-well plates pre-coated with IFN-γ antibodies (clone 1-D1K, Mabtech AB, Nacka Strand, Sweden). The cells were then separately stimulated with each of the 46 or 47 peptides from the corresponding peptide pool at a concentration of 10 µg/ml for 18–20 h at 37°C and 5% CO₂. Anti-CD3-stimulated cells served as a positive control, and unstimulated cells in R10 medium served as a negative control.
After a washing step, IFN-γ was detected with a biotinylated anti-IFN-γ antibody (clone 7-B6-1; Mabtech AB, Nacka Strand, Sweden), alkaline phosphatase-conjugated streptavidin, and a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) substrate solution. Results were considered positive if a single peptide well showed at least three times the number of IFN-γ spots compared to the corresponding control well.