Measuring SIVmac239-specific Immune Responses

We evaluated SIVmac239-specific immune responses through the IFN-γ-mediated ELISpot assay as we previously reported (52 (link)). Briefly, a 96-well PVDF plate (Merck Millipore, Cat. No. MSBVS1210) was coated with monoclonal coating antibodies (U-CyTech, Cat. No. CT605-10) overnight at 4°C. PBMCs were isolated through density gradient centrifugation by Lymphoprep (Axis-Shield, Cat. No. 1114547). The plate was washed three times with DPBS (BasalMedia, Cat. No. B210KJ) and 200 µL per well of R10 medium (RPMI 1640, 10% fetal bovine serum, 1% penicillin‒streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, and 10 mM HEPES) were added to block the plate for 2 h. PBMCs were diluted with R10 medium, and 300,000 PBMCs were added to each well. The SIVmac239 peptide stocks were diluted 20 times by R10, and 10 µL of each peptide was added to the corresponding well. After incubation for 24 h, the plate was washed, and biotinylated monoclonal detection antibodies (U-CyTech, Cat. No. CT605-10) were added. The next morning, streptavidin-AKP (BD PharMingen, Cat. No. 554065) was added and incubated for 2 h at 37°C. The 1-STEP NBT/BCIP (Thermo Scientific, Cat. No. 34042) was then added and incubated for 10 min at 37°C. The plate was washed and dried before being read by an enzyme-linked immunospot imager (Bioreader6000, BIOSYS, Germany).