Anti ifn γ capture monoclonal antibody

PBMCs from participants were obtained from whole blood by Ficoll-Hypaque density gradient centrifugation (Ficoll-Paque Plus; Amersham Biosciences) and then resuspended in Lympho-Spot medium (U-CyTech Bioscience, The Netherlands). Then, 2 × 10⁵ cells were seeded in duplicates in 96-well plates (MultiScreen-IP; Millipore) precoated with anti-IFN-γ capture monoclonal antibody (eBioscience). Cells were stimulated with the peptide pool (ESAT-6 amino acids [aa] 21 to 40, aa 51 to 70, and aa 71 to 90 and CFP-10 aa 21 to 40, aa 51 to 70, and aa 66 to 85) for 24 h at 37°C with 5% CO₂ as described previously (37 (link)). PBMCs in medium alone or stimulated with phytohemagglutinin (Sigma) at 2.5 μg/ml were used as negative or positive controls, respectively. Biotinylated anti-IFN-γ detection monoclonal antibody (eBioscience) was added for 4 h, followed by the addition of streptavidin-alkaline phosphatase conjugate (Pierce Biotechnology) for 1 h. After a washing step, the nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate; Sigma) chromogenic substrate was added. The individual spots were counted by use of an automated image analysis system ELISPOT reader (BioReader 4000 Pro-X; Biosys, Germany).