ELISPOT assay

MultiScreen Filter PVDF Immobilon plates (Merck Millipore, Darmstadt, Germany) were treated with 35% ethanol for 1 min prior to being washed five times with sterile water. The wells were then coated overnight at 4 °C with 15 µg/mL of monoclonal antibodies of anti-human IgG (clones MT91/145; Mabtech), 10 µg/mL of recombinant PvMSP1P-19 or PvMSP1 antigen, or 5 µg/mL of tetanus toxoid (TT) antigen (Merck Millipore, Darmstadt, Germany) diluted in sterile PBS. The plates were washed with 200 µL of sterile PBS five times and blocked with 200 µL of R10 for at least 30 min at 37 °C in 5% CO₂. Both the stimulated and non-stimulated cells were harvested from the culture plates, counted and seeded in duplicate to yield 5 × 10⁴ cells per anti-human IgG-coated well and 1 × 10⁶ cells per specific antigen-coated well. After 24 h of incubation at 37 °C and 5% CO₂, the plates were then washed with PBS five times. Then, 1 µg/mL of detection monoclonal antibodies MT78/145 (Mabtech) diluted in PBS-0.5% FBS were added and incubated for 2 h at room temperature. After five washes with PBS, the immobilized IgG was labelled using streptavidin–horseradish peroxidase (HRP)-conjugated polyclonal goat anti-human IgG (Mabtech) diluted to 1:1000 in PBS-0.5% FBS; Mabtech) and incubated for 1 h at room temperature. Following a thorough washing with PBS, tetramethylbenzidine (TMB) substrate (Mabtech) was added. The filtered plates were rinsed with deionized water after distinct spots emerged. Antigen-specific MBCs were expressed as spot-forming cells (SFC) in the wells. The plates were then left to dry and stored to protect them from light until they could be analysed with a Bioreader 5000 Pro-F gamma ELISPOT Reader (BioSys GmbH, Karben, Germany). PBMCs from P. vivax subjects that were cultured without stimulation and then incubated overnight with PvMSP1P-19 antigens were used as negative controls. The positive ELISPOT response was defined when spots were detected in duplicate wells and the total spots in the specific antigen-coated well reached at least twice the number of spots detected in the negative controls.