Anti mouse ifn γ clone an18

Elispot plates (MSIPS 4510, Millipore) were coated with 5 µg/ml of anti-mouse IFN-γ (clone AN18, MabTech) capture antibody in the dark at 4 °C overnight. Plates were blocked with R10 complete medium at 37 °C for a minimum of 2 hours. Mouse PBMCs or splenocytes were incubated with 5GHPV3 peptide pools or individual peptides at a final concentration of 10 µg/ml. PMA/ionomycin at 5 μg/ml, and R10 (0.1% DMSO) were used as positive and negative control, respectively. Plates were incubated for 16–18 hours at 37 °C, after which a biotinylated anti-mouse IFN-γ detection antibody was added for 2 hours at room temperature. Streptavidin alkaline phosphatase polymer (Mabtech) was added for 1 hour at room temperature and the plates developed with 50 µL of 1-Step NBT/BCIP Substrate Solution (ThermoFisher Scientific) for 5 to 10 minutes until visible spots were observed on the membrane. Spot-forming units were counted using an automated reader (AID). The same procedure was used for human IFN-γ Elispot assays, apart from the use of anti-human IFN-γ coating antibody (clone 1-D1K, MabTech) and a biotinylated anti-human IFN-γ detection antibody (clone 7-B6-1, MabTech). The final peptide concentration for all the pools was 2 µg/ml. The assay cut-off for defining a positive response was 25 SFU/million (derived from mean + 3 SD of mock-stimulated well values).

Splenocytes were stimulated with a panel of 11 overlapping 15–19 mer peptides (NIHAIDS reagent program) spanning the entire Tat proteinas we described28 (link)29 (link). Briefly, multiscreen-IP HTS plates (Merck Millipore, Germany) were coated with anti-mouse IFN-γ (clone AN18, MabTech, Sweden) and secreted IFN-γ detected with anti-mouse IFN-γ -biotin (clone R4-6A2, MabTech).Phytohemagglutinin and un-stimulated splenocytes were used as positive and negative controls, respectively. Spots were counted using an ELISpot reader (AID GmbH, Germany). The number of spots in unstimulated splenocytes was subtracted from the number in peptide-stimulated cells to generate the net number of Tat-specific spot forming units (SFUs).

ELISpots were performed to determine the breadth and magnitude of HIV-specific CMI in splenocytes from vaccinated mice. A panel of 15–19 mer overlapping peptide pools spanning the entire Gag and Tat proteins was obtained from the NIH AIDS reagent bank (Germantown, MD, USA). Mouse interferon (IFN)-γ ELISpot was performed on RBC-depleted splenocytes that were re-stimulated for 36 h with 4 μg/ml of 5 Gag peptide pools or a Tat peptide pool as we described previously24 (link)38 (link). Briefly, multiscreen-IP HTS plates (Millipore) were coated with anti-mouse IFNγ (clone AN18, MabTech) and secreted IFNγ detected with anti-mouse IFNγ-biotin (clone R4-6A2, MabTech). Developed spots were counted automatically using an ELISpot reader (AID GmbH, Germany). The number of spots (spot forming units-SFUs) in unstimulated splenocytes was subtracted from the number in peptide-stimulated cells to generate the net Gag or Tat response.