Quantifying Th1 Immune Memory with IFN-γ ELISpot

ELISpot This assay is designed to determine the proportion of T cells that release IFN-γ after stimulation with SLA in order to quantify the level of stimulation of a specific Th1 polarity immune memory response. It was performed in a manner similar to that previously described [19 (link),27 (link)]. Heparinized blood samples were fractionated by centrifugation over lymphocyte separation medium. The PBMCs obtained were incubated at a density of 10⁶ cells/mL for 3 days in multiscreen HTS filter plates (Millipore, Billerica, USA) previously coated with canine IFN-γ capture antibody (R&D System, Minneapolis, USA), in presence of 10 μg/mL ConA, or 10 μg/mL SLA antigens, or with medium alone, in a humidified 37 °C CO2 incubator. The quantity of IFN-γ was revealed with a specific biotinylated antibody and incubation with Streptavidin-AP and the BCIP/NBT Chromogen (R&D System, Minneapolis, USA). The number of specific spots was determined by an automated ELISpot reader. ConA was used as a positive control and the medium alone was used as a negative control. The data presented are the number of spots per 2 × 10⁵ cells after stimulation with SLA minus the equivalent value obtained with the negative control using medium alone.