Immobilon p pvdf plates

Immobilon-P (PVDF) plates (Millipore, Bedford, MA, USA) were activated using 35% ethanol for 1 min and coated with 4 μg ml⁻¹ of purified mouse anti-IL-17A and anti-IFN-γ (both from BD Biosciences, San Diego, CA, USA) overnight at 4 °C. Plates were blocked with 1% BSA, equal number of LN or spleen cells were loaded per well and incubated with increasing doses of MOG_35–55 for 36 h at 37 °C. Plates were then washed and biotinylated anti-IFN-γ and anti-IL-17A (2 μg ml⁻¹) were added and incubated overnight at 4 °C. Afterwards, the plates were again washed and incubated at room temperature for 2 h with alkaline phosphatase (Sigma, St Louis, MO, USA). Plates were then developed in BCIP/NBT (Sigma) solution. A computer-assisted ELISPOT Image Analyzer (Cellular Technology Limited, Cleveland, OH, USA) was used to count the spots.