Biotinylated anti human ifnγ detection antibody

For ELISPOT assays, MoDC/T cell or MoDC/PBMC co-cultures were performed in 96-well MultiScreen HTS IP plates (0.45 μm, clear, Merck Chemicals GmbH, Darmstadt, Germany), coated with anti-IFNγ capture antibody (BD Biosciences, Heidelberg, Germany) overnight at 4°C. Following blocking for two hours at room temperature with culture medium, cells were seeded and stimulated as described above. After 16 h incubation, cells were discarded and plates washed in sterile water and PBS / 0.05% Tween20 (Sigma-Aldrich Chemie GmbH, Munich, Germany). Biotinylated anti-human IFNγ detection antibody (BD Biosciences, Heidelberg, Germany) was added in PBS / 10% FCS and plates incubated for two hours. After washing in PBS / 0.05% Tween20 Alkaline Phosphatase (AP)-conjugated Streptavidin (BD Biosciences, Heidelberg, Germany) was added 1:1000 in PBS/10% FCS followed by incubation for one hour. Development of the plate was performed with the AP conjugate substrate kit (Bio-Rad Laboratories GmbH, München, Germany); the reaction was stopped with water and the plate dried overnight.
FluoroSpot assay was carried out using the MabTech Kit (IFNγ/IL-13, MabTech, Stockholm, Sweden) following the provided protocol.
Spots and enzymatic activity were quantified with an iSpot FluoroSpot Reader System (AID, Strassberg, Germany).

Human PBMCs were prepared from healthy donors under informed consent, with ethical approval under Research Tissue Bank ethics number RTB 16/EE/0334. PBMCs were isolated from leukocyte cones using a polysucrose (Ficoll) gradient and maintained in RPMI 1640 (supplemented with 10% v/v heat-inactivated FBS, 1% v/v Penicillin/Streptomycin) at 37°C in a 5% CO₂, humidified atmosphere. PBMCs were stimulated with 5 ng/ml human IL-12, 0.1 ng/ml oxidation resistant human IL-33 (human IL-33^112–270, C208S, C227S, C232S and C259S) and HpBARI, HpBARI_Hom2 or control protein (HpAChE, Hp_I12803_IG04747_L2174). Cells were incubated for 44 hr. Media supernatants were collected and soluble IFN-γ was assessed by ELISA, using anti-human IFN-γ capture antibody (Pharmingen, #551221), biotinylated anti-human IFN-γ detection antibody (Pharmingen, #554550) and DELFIA Europium visualisation (PerkinElmer). Data were analysed using Graphpad Prism software. IC50 values were determined by curve fitting using a four-parameter logistic equation, and a mean value taken of the response of 5 donors. Pooled data from 3 of the 5 donors, where identical titrations of protein were carried out, are shown in Figure 8E.