Monoclonal anti bovine ifnγ antibody

IFNγ ELISpot assays were conducted as described in [36 (link)]. Briefly, a monoclonal anti-bovine IFNγ antibody (Serotec, Oxford, UK, cat.no. MCA1783) was incubated overnight at 4 °C on ELISpot plates (Millipore, Billerica, MA, USA) and then blocked with RPMI containing 10% heat-inactivated FBS for 2 h at 37 °C. Peptides were added at 1 μM concentration and CTL lines at 2.5 × 10⁴ cells per well. The plates were incubated at 37 °C for 20 h. Release of IFNγ was monitored with primary rabbit polyclonal anti-bovine IFNγ antibody (Sigma–Aldrich, St. Louis, MO, USA) and secondary AP-conjugated monoclonal anti-rabbit antibody (Sigma–Aldrich, St. Louis, MO, USA, cat # A2556). Development of plates was done by addition of the substrate solution Sigma Fast (BCIP/NBT, Sigma–Aldrich, St. Louis, MO, USA).

Interferon (IFN)-gamma (γ) enzyme-linked immunospot (ELISpot) assay was performed as previously described.^{33 (link)} Briefly, CD8⁺ cells from whole Ficoll-purified PBMC were isolated using the MACS Cell Separation system (with anti-mouse IgG MicroBeads, Miltenyi Biotec, cat # 130-048-401) following the manufacturer’s instructions. A monoclonal anti-bovine IFNγ antibody (Serotec, Oxford, UK, cat. no. MCA1783) was incubated overnight at 4 °C on ELISpot plates (Millipore, cat # MAIPN4550, Billerica, MA, USA) and then blocked with RPMI containing 10% heat-inactivated FBS for 2 h at 37 °C. Peptides were added at 1 μM concentration and CD8-positive cells at varying concentrations ranging from 3.125 × 10⁴ to 2.5 × 10⁵ cells per well. The plates were incubated at 37 °C for 20 h. Release of IFN-γ was monitored with primary rabbit polyclonal anti-bovine IFN-γ antibody (Sigma-Aldrich, St. Louis, MO, USA) and secondary AP-conjugated monoclonal anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA, cat # A2556). Development of plates was done by addition of the substrate solution Sigma Fast (BCIP/NBT, Sigma-Aldrich, cat # B5655-25TAB, St. Louis, MO, USA).