Extravidin phosphatase

ELISA α-Syn protein quantification was performed as previously described [38 (link), 39 (link)]. 384-well MaxiSorp plates (Nunc, Inc) were coated with capturing antibody (α-Syn, BD Biosciences, 610787, RRID:AB_398108) diluted 1:500 in coating buffer (NaHCO₃ with 0.2% NaN₃, pH9.6) overnight at 4 °C. Following 3 washes with PBS/0.05% Tween 20 (PBS-T), plates were blocked for 1 h at 37 °C in blocking buffer (1.125% fish skin gelatin; PBS-T). After 3 washes, samples were loaded in duplicates and incubated at RT for 2 h. Biotinylated hSA4 antibody (in-house antibody, RRID:AB_3095299) was generated using 200 μg Sulfo-NHS-LC Biotin (Pierce, Thermo Fisher Scientific), diluted 1:200 in blocking buffer and added to the plate for 1 h at 37 °C. Following 5 washes, ExtrAvidin phosphatase (Sigma, E2636) diluted in blocking buffer was applied for 30 min at 37 °C. Color development was carried out by using fast-p-nitrophenyl phosphate (Sigma, N1891) and monitored at 405 nm every 2.5 min for up to 60 min. Saturation kinetics were examined for identification of time point(s) where standards and sample dilutions were in the log phase. 10.17504/protocols.io.261gedywyv47/v1.

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.