Capture antibodies against γifn

ELISPOT plates (Millipore, Bedford, MA) were pre-coated with 5 μg/ml of capture antibodies against γIFN (Mabtech, Sweden) in PBS and stored overnight at 4°C. The responding cells, CD4 Tmems and CD8 Tmems, separated from fresh PBMCs were co-cultured with an equal number of irradiated donor PBMCs as stimulating cells (1.5 × 10⁵ cells/well), or unstimulated in medium alone, or with PHA at 1 μg/ml (Sigma). After 44 hours incubation at 37°C, the plates were washed and biotinylated detection antibodies (Mabtech, Sweden) were added (4°C OVN). After 5 washes with PBS, streptavidin-horseradish-peroxidase conjugate in PBS BSA 0.5% (Dako, Glostrup, Denmark) was added for 2 hrs at room temperature, followed by 5 washes. Finally, 50 μl/well of tetramethylbenzidine (TMB) liquid substrate (Sigma-Aldrich) was added and incubated for 30 min in the dark. The resulting spots were counted with an ELISPOT image analyzer (CTL Inc., Cleveland, OH), as described elsewhere (13 ).

ELISPOT plates (Millipore, Bedford, MA) were precoated with 5 µg/mL of capture antibodies against γIFN (Mabtech, Sweden) in PBS and stored overnight at 4°C. Purified recipient’s T cells were co-cultured with an equal number of irradiated donor PBMCs as stimulators (1.5 × 10⁵ cells/well), or unstimulated in medium alone, or with PHA at 1 µg/mL. After 44h of incubation, biotinylated detection antibodies (Mabtech, Sweden) were added and the spots were revealed and counted, as described elsewhere (8 ).