Elispot aec substrate

Human donor HLA-A02 PBMCs were acquired from Hemacare. Viral
peptides chosen for T-cell memory and reactivity were the CEF Peptide Pool
(Anaspec), CMVpp65 (495–503), EBV BMLF1 (280–288) and the CEF Scramble negative
control peptide pool (JPT), as well as PMA/Ionomycin as a positive control.
ELISpot was performed with the VECTASTAIN ABC HRP Kit (Vector Laboratories)
according to the manufacturer’s instructions, using a primary anti-human-IFNg
(Mabtech) monoclonal antibody and a secondary IFNg-Biotin antibody on 96-well
ELISpot plates (Millipore). ELISPOT AEC Substrate (BD Biosciences) was used to
resolve spots on membranes and IFNg⁺ cells were
evaluated in a blinded fashion (ZellNet Consulting, Inc.) using an ELISpot
reader (KS ELISpot reader, Zeiss) with KS ELISpot software version 4.9.16. The
plate evaluation process, including the setup of optimal reading parameters,
followed the International Guidelines on ELISpot plate
evaluation^{32 (link)}.

Ninety-six-well filtration plates (Millipore, MA, USA) were pre-coated with anti-mouse IFN-γ antibody (5 μg/ml; BD Biosciences) overnight at 4 °C. After the plates were washed four times with PBS and then blocked with RPMI 1640 medium (Gibco), CD8⁺ T cells (1 × 10⁵ cells in 100 μl of medium) were added to each well of the filtration plates along with UV-inactivated influenza virus (1 TCID₅₀) [45] in 100 μl of medium). The plates were incubated for 72 h, washed with PBS containing 0.5 % Tween-20 (PBS-T), and incubated with biotin-conjugated anti-mouse IFN-γ antibody (1 μg/ml) for 2 h at room temperature. After 5 washes with PBS-T, diluted horseradish peroxidase (HRP)-conjugated streptavidin antibody (1:200; BD Biosciences) was added to the each well and incubated at room temperature for 1 h. The cells were washed 5 times with PBS-T; ELISPOT AEC substrate (100 μl; BD Biosciences) was added to develop the reaction, and then the reaction was stopped by washing the cells with double-distilled water. The spots were counted automatically by using the ELISPOT CTL-ImmunoSpot S5 UV Analyzer (Cellular Technology; OH, USA).