Anti human ifn γ coating antibody clone 1 d1k

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).

PBMCs (1-2×10⁶) were cultured at 37°C and 5% CO2 for 10 days in R10 medium (RPMI with 10% fetal calf serum) plus IL-7 (25 ng/ml) with HPV peptides identified from the cervical cancer panel (4 μg/ml) or a pool of irrelevant FEC peptides (2 μg/ml). Cells were supplemented with 1,800 U/ml IL-2 on days 3 and 7 and additional R10 on day 7. Cells were collected on day 10, washed four times with PBS and rested at 37°C and 5% CO2 for 29–34 h in fresh R10. Elispot plates (MSIPS 4510, Millipore) were coated with anti-human IFN-γ coating antibody (clone 1D1K, MabTech) in the dark at 4°C overnight. Plates were blocked with R10 complete medium at 37°C for a minimum of 2 h. Cells (1 × 10⁵/well) were added and incubated with HPV peptides (2ug/ml) or FEC (4ug/ml). PHA at 5 μg/ml and R10 (0.1% DMSO) were used as positive and negative control, respectively. Plates were incubated for 16–18 h at 37°C, after which a biotinylated anti-human IFN-γ detection antibody (clone 7-B6-1, MabTech) was added for 1 h at room temperature. Plates were then washed 6 times and ABC complex added for 1 h at room temperature followed by the addition of AMEC Complex Buffer. Spot-forming units were counted using an automated reader (AID). The magnitude of response was defined as the number of spot-forming units (SFU) per million PBMCs following background subtraction.