Immobilon p plates

For ELISA, 96-well plates (2595; Costar) were coated with 2 μg/mL NP₃₀-BSA or 10 μg/mL OVA (Biosearch Technologies and Sigma-Aldrich) in PBS overnight. Plates were washed twice (0.5% BSA, 0.1% Tween 20 in PBS), blocked for 1 hr (0.5% BSA in PBS), and washed twice, and serially diluted samples were addedfor 1 hrat RT. Plates werewashedthree times and horseradish peroxidase (HRP)-conjugated detection Abs for IgG (Bethyl Laboratories) were added for 1 hr, washed three times, and tetramethylbenzidine substrate was added (BD Biosciences). Reaction was stopped using 2 N H₂SO₄ and read at 450 nm. Ab quantification was calculated based on NP- or OVA-specific monoclonal standards (H33lγ1 or OVA-14) and reported as relative units (RU). For ELISPOTs, Immobilon-P plates (Millipore) were coated overnight at 4°C with 2 μg/mL NP₃₀-BSA or 10 μg/mL OVA in PBS. Plates were washed twice then blocked for 2 hr. BM cells were isolated; RBCs were lysed with ACK, and then incubated for 3 hr at 37°C in RPMI. The plates were washed three times, and then incubated with alkaline phosphatase-conjugated Abs to IgG (SouthernBiotech). Plates were washed five times and developed using NBT reagent (Sigma-Aldrich).

Porcine PBMCs were isolated from blood samples collected in Vacutainer tubes EDTA-K2, diluted 1:1 in PBS and then used to obtain PBMC by density-gradient centrifugation with Histopaque 1077 (Sigma) and Leucosep tubes (Greiner Bio-One) as described (28 (link)). For the IFN-γ ELISPOT assay 2.5 × 10⁵ PBMCs were shed in triplicate wells of Immobilon-P plates (Merck Millipore) coated as reported (19 (link)) and in vitro stimulated with 50 μg/ml of their respective immunogenic peptides. As positive or negative controls, cells were incubated with 10 μg/ml of phytohaemagglutinin (Sigma) or only with medium, respectively. After 48 h at 37°C and 5% CO₂, plates were washed and incubated with a biotinylated mouse anti-pig IFN-γ (clone P2C11, BD) followed by streptavidin:HRP (BD). The frequency of peptide-specific T-cells was expressed as the mean number of spot-forming cells/10⁶ PBMCs, with background values (number of spots in negative control wells) subtracted from the respective counts of stimulated cells. These experiments were performed using outbred domestic pigs with different individual genetic backgrounds. In any case, the levels of animal-to-animal variation did not exceed those observed in other related studies (11 (link)).

Porcine peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Histopaque-1077 (Sigma, St. Louis, MO, USA) and cryopreserved prior to assay. Vials were defrosted and resuspended in complete RPMI 1640 and incubated overnight at 37 °C, 5% CO₂. Cell counting and viability were tested by Trypan blue staining. For the IFN-γ ELISPOT assay 2.5 × 10⁵ PBMCs were shed in triplicate wells of Immobilon-P plates (Merck Millipore, Burlington, MA, USA) coated with 5 µg/mL of anti-pig IFN-γ antibody (clone P2G10, Becton Dickinson, Franklin Lakes, NJ, USA). For in vitro antigen recall, PBMCs were stimulated with 25 μg/mL of the peptide used for pig immunization [49 (link)]. As positive control, PBMCs were incubated with 5 μg/mL of phytohaemagglutinin (Sigma) using cells incubated without antigen as negative control. After 48 h at 37 °C, 5% CO₂, plates were washed and incubated with 2 µg/mL of biotinylated anti-mouse IFN-γ antibody (clone P2C11, BD) and HRP-streptavidin (BD). Antibody was visualized with 3-amino-9-ethyl carbazole (BD). The frequency of peptide-specific T cells in was expressed as the mean number of spot-forming cells/10⁶ PBMCs, with background values (number of spots in negative control wells) subtracted from the respective counts of stimulated cells.

Porcine peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). Cell counting and viability were tested by trypan blue staining. For the IFN-γ ELISPOT assay 2.5 × 10⁵ PBMCs were shed in triplicate wells of Immobilon-P plates (Merck Millipore, Madrid, Spain) coated with 5 µg/mL of anti-pig IFN-γ antibody (clone P2G10, BD Biosciences, San Agustín de Guadalix, Madrid, Spain). For in vitro antigen recall, PBMCs were stimulated with 50 μg/mL of the peptide used for pig immunization [38 (link)]. As positive control, PBMCs were incubated with 10 μg/mL of phytohaemagglutinin (Sigma-Aldrich, St. Louis, MO, USA) using cells incubated without antigen as negative control. After 48 h at 37 °C—5% CO₂, plates were washed and incubated with 2 µg/mL of biotinylated anti-mouse IFN-γ antibody (clone P2C11, BD Biosciences) and HRP-streptavidin (BD Biosciences). Antibody was visualized with 3-amino-9-ethyl carbazole (BD Biosciences). The frequency of peptide-specific T-cells was expressed as the mean number of spot-forming cells/10⁶ PBMCs, with background values (number of spots in negative control wells) subtracted from the respective counts of stimulated cells.

Quantification of IFN-γ secreting cells from immunized and control animals was performed using an ELISPOT assay. Briefly, 2.5 × 10⁵ PBMCs were shed in triplicate wells of Immobilon-P plates (Merck Millipore) coated as reported (18 (link)) and in vitro stimulated with 50 µg/ml of their respective dendrimers or with T or B peptides. As positive or negative controls, cells were incubated with 10 µg/ml of phytohaemagglutinin (Sigma) or only with medium, respectively. After 48 h at 37°C and 5% CO_2, plates were washed and incubated with a biotinylated mouse anti-pig IFN-γ antibody (clone P2C11, BD) followed by HRP-streptavidin (BD). The frequency of peptide-specific T-cells was expressed as the mean number of spot-forming cells/10⁶ PBMCs, with background values (number of spots in negative control wells) subtracted from the respective counts of stimulated cells. These experiments were performed using outbred domestic pigs with different individual genetic backgrounds. In any case, the levels of animal-to-animal variation did not exceed those observed in other related studies (11 (link)).