Il 17 mab

Ninety-six-well multiScreen filter plates (Millipore, USA) were pre-wetted with 35% ethanol then washed twice with PBS before coating. Plates were coated at 4 °C overnight with 10 μg/ml PR8 for B-cell ELISPOTs or 5 μg/ml anti-mouse IL-2, IL-4, or IFN-γ (all from BD Biosciences) or IL-17 mAb (BioLegend, San Diego) for T-cell ELISPOTs. The plates were washed with PBS, then blocked with RPMI 1640 + 10% FCS. A total of 4 × 10⁵ splenocytes and 5 μg/ml PR8 were added in triplicates before the plates were incubated at 37 °C for 24 h (B-cell ELISPOT) or 48 h (T-cell ELISPOT). The plates were washed 3 times with PBS-Tween 20 and 2 μg/ml detection antibodies in PBS + 10% FCS were added and incubate for overnight at 4 °C. After discarding detection antibodies, plates were washed 3 times with PBS/Tween 20 and incubated with 1:200 dilution of HRP-Streptavidin (BD Biosciences) for 1 h at RT. After washing spots were visualized using AEC substrate (BD Biosciences). The plates were washed twice with water, dried, and analyzed by Immunospot ELISPOT system (Cellular Technology, Shaker Heights, OH, USA).