ELISpot Assay for PfCelTOS Antigen

ELISpot was performed as previously described [35 (link)] and according to the manufacturer’s instructions (R&D Systems, SEL485 and SEL404, Minneapolis, MN, USA). Briefly, rPfCelTOS (10 µg/mL) was used as the stimulating antigen. Hamster anti-mouse CD3e (1 µg/mL [BD Biosciences, 553057, San Jose, CA, USA]) was used as a positive control for cell stimulation. The negative control was culture media in place of a stimulating antigen. Splenocytes were plated at 2 × 10⁵ cell/well. Plates were incubated for 48 h at 37 °C with 5% carbon dioxide. Spot counting was performed using an AID ELISpot Reader (Autoimmun Diagnostika, Strassberg, Germany).