Quantifying IFN-γ-secreting T-cells by EliSpot

The number of IFN-γ-secreting T-cells was determined by the Porcine IFN-γ EliSpot BASIC kit (3130-2A, MabTech, OH, USA) using PBMCs and splenocytes pulsed with peptides as previously described (54 (link), 69 (link)). In brief, each sample was assayed in triplicate in MultiScreen-HA 96-well-plates (MAIPS4510, Millipore, MO, USA) with 2.5 x 10⁵ cells/mL cells pulsed with 2.5 μg/mL of each peptide in cRPMI 1640 media. Peptide screening was carried out using four pools [A-D] containing 18 9-mer peptides and a final pool [E] contained the remaining 16 peptides (Table 2). Reactive pools were then tested at the individual peptide level at the same concentration indicated earlier. For each test, positive and negative controls were Phytohemagglutinin (PHA) mitogen at a concentration of 5 μg/mL and media alone, respectively. After a 48-h incubation at 37°C in 5% CO₂ atmosphere, plates were developed as per the MabTech protocol, the membranes air-dried in the dark, and spots were detected using EliSpot reader (MabTech, OH, USA) and AID software (version 3.4; AutoImmun Diagnostica, Strasburg, Germany). Data are presented as Spot Forming Cells (SFC)/10⁶ PBMCs or splenocytes based upon the mean number of peptide-specific IFN-γ producing cells after subtracting the negative control mean counts as background.