Quantifying HIV-Specific T-Cell Responses by ELISPOT

Peripheral blood mononuclear cells (PBMC) were resuspended at 1×106/mL in RPMI supplemented with 10% FBS (R10) and plated 200 μL per well in Immobilon-P 96-well microtiter plates (Millipore) pre-coated with 2 μg/mL anti-IFN-γ (clone DK1, Mabtech). Individual HLA-optimal HIV peptides matched to each subject’s HLA genotype were added at 1 μM and incubated at 37°C overnight. Negative control wells did not receive peptide and positive control wells were treated with 1 μg/mL anti-CD3 (clone OKT3, Biolegend) and 1 μg/mL anti-CD28 (clone CD28.8, Biolegend) antibodies. ELISOPT assay was performed using manufacturer’s protocol with anti-IFN-γ (clone 1-DK1, Mabtech) capture, biotinylated anti-IFN-γ (clone B6–1, Mabtech) detection, Streptavidin-ALP (Mabtech) and AP Conjugated Substrate (BioRad) followed by disinfection with 0.05% Tween-20 (Thermo Fisher) and analysis using S6 Macro Analyzer (CTL Analyzers). Responses greater than 10 spots per well and 3-fold above negative controls were scored as positive.^{87 (link),88 (link)}