Anti ifn γ capture antibody

The ELISpot-technique was applied for sensitive detection of IFNγ-secreting NK cells. First, a Multiscreen 96-well filtration plate (Merck Millipore) was activated with 35% ethanol and coated with anti-IFNγ capture antibody (200 µg/ml, clone 1-D1K, mIgG1, k, Mabtech). After incubation at 4 °C overnight, the plates were blocked with 200 µl of supplemented complete RPMI-1640-media for 2 h at 37 °C. After the washing step isolated NK cells were coincubated with IGROV1 cells in 1:1 ratio and either with or without ascites samples (ratio 1:4). For ADCC conditions, Cetuximab 1 µg/ml (Erbitux, 5 mg/mL, Merck (Serono) was added. After incubation for 24 h at 37 °C and 5% CO₂ plates were washed in the ELISA-washer (PBS / 0,05% Tween-20.) Biotinylated anti-IFNγ detection antibody (200 µg/ml, clone 7-B6-1, mIgG1, k, Mabtech) was added in 2 µg/ml PBS and 1% BSA. The plates were incubated for 2 h at 37 °C, washed and incubated with 50 µl ExtraAvidin alkaline phosphatase (1:1000 diluted in PBS / 1% BSA, Sigma-Aldrich) at room temperature for 2 h. After the washing steps, 75 µl of the ELISpot substrate BCIP/NBT (Roche) was added and incubated for 5–10 min. Developed cytokine spots were measured using AID Classic ELISpot Reader and the results were analyzed with AID ELISpot 7.0 software.

The ELISpot-technique was applied for sensitive detection of IFNγ-secreting NK cells. First, Multiscreen 96-well filtration plate (Merck Millipore) was activated with 35% ethanol and coated with anti-IFNγ-capture antibody (200µg/ml, clone 1-D1K, mIgG1, k, Mabtech). After incubation at 4°C overnight the plates were blocked with 200µl of supplemented complete RPMI-1640-media for 2h at 37°C. After washing step isolated NK cells were seeded in triplicates and treated with different nanoparticle types. NK cells treated with PMA (50ng/ml, Sigma-Aldrich) and Ionomycin (1µg/ml, Sigma-Aldrich) were included for positive control. Untreated NK cells were used as negative control. After incubation with 12,5 µl of calcium phosphate nanoparticles for 24 h at 37°C and 5% CO2 plates were washed in the ELISA-Washer (PBS/0,05% Tween-20.) Biotinylated anti-IFNγ-detection antibody (200µg/ml, clone 7-B6-1, mIgG1, k, Mabtech) was added in 2µg/ml PBS and 1% BSA. The plates were incubated for 2 h at 37°C, washed and incubated with 50µl ExtraAvidin alkaline phosphatase (1:1000 diluted in PBS/1% BSA, Sigma-Aldrich) for 2h at the room temperature. After washing steps 75µl of the ELISpot substrate BCIP/NBT (Roche) was added and incubated for 5-10 minutes. Developed cytokine spots were measured using AID Classic ELISpot Reader and the results were analyzed with AID ELISpot 7.0 software.

Ninety-six well filter plates were pre-coated with anti-IFN-γ capture antibody (MabTech). Spleens were isolated from mice 2 weeks after the final immunization. After processing the spleens to obtain a single-cell suspension, 2 × 10⁵ cells were added to the blocked plates. Cells were stimulated with overlapping 15mer peptide pools for WT BG505 gp160 (5 ug/ml per peptide—GenScript). Media alone and concanavalin A (Invitrogen) were used as negative and positive controls respectively. After 18 h of stimulation at 37 °C, the plates were washed, and the detection antibody (R4-6A2-biotin) was added for 2 h at RT. Plates were then washed and the Streptavidin-ALP antibody (1:1000) was added for 1 h at RT. Plates were then developed using the BCIP/NBT-plus for 10 min. Plates were then scanned and counted using CTL-ImmunoSpot^® S6 FluoroSpot plate reader (Cellular Technology Limited CTL).