Biotinylated anti ifn γ antibody

A multiscreen, 96-well filtration plate (Millipore) coated with antihuman IFN-γ antibody (Human IFN-γ ELISpot kit; Mabtech (Cincinnati, OH, USA)) was used. CD3⁺ T cells from patients or healthy donors were isolated (CD3 Microbeads; Miltenyi). CD3⁺ T cells (1 × 10⁶ in 100 μL volume) were added to each well alone, or co-cultured with autologous MoDCs (1 × 10⁵/well) with or without CMV peptide (PepTivator CMV pp65, Miltenyi), with medium alone as a negative control, or with PHA (Sigma–Aldrich (St. Louis, MO, USA)) as a positive control. All ELISpot assays were carried out in triplicate. After 24 h incubation at 37 °C/5% CO2, cells were removed by washing the plates four times with PBS containing 5% Tween 20 and twice with PBS. Fifty microliters of biotinylated anti-IFN-γ antibody was added (1:1000 dilution; Mabtech) and incubated for 3 h at room temperature. The ELISpot plate was washed an additional six times with PBS/Tween 20 and incubated for 2 h with streptavidin-ALP substrate (Mabtech) followed by the addition of an alkaline phosPHAtase conjugate substrate (Mabtech). The resulting spots were counted semi-automatically with Bioreader 4000 (Pro-X, BIOSYS GmbH, Werner Freber Germany). Results were expressed as number of cells secreting IFN-γ per well.