Quantifying IFNγ and IL-17A Responses

The IFNγ and IL-17A ELISPOT assay was performed as described by Maggioli et al.^{39 (link),50} . Briefly, 96-well ELISPOT plates (Millipore) were coated at 4 °C overnight with an anti-bovine IFNγ capture mAb or IL-17A capture mAb (both from Kingfisher Biotech, Inc., St. Paul, MN), followed by a blocking step in cRPMI, for 2 h at 37 °C, 5% CO₂. Fresh isolated PBMC or long-term cultured cells (2 × 10⁴/well) were added to ELISPOT plates and stimulated with either PPD-b (5 μg/ml), rTB10.4 and rAg85A (1 μg/ml each), ConA (5 µg/ml) or medium alone. Plates were incubated 20 h (IFNγ) or 48 h (IL-17A) at 37 °C, 5% CO₂. Spot forming cells (SFC) were detected following the Vectastain ABC-AP Kit (Vector Laboratories, Burlingame, CA) standard procedures.