Innoscan scanner

Glass slides were coated with amine solution as described in a previous report^{25 (link)}. Evaluation of the acquired immune response was assessed in 32 pairs of vivax sera samples (D0, D28, and 1 yr). Sera pooled from eight vivax malaria patients or eight knowlesi malaria patients were used to evaluate cross-immunoreactivity. The crude rPvAARP-FL and rPkAARP-FL proteins were spotted in duplicate, incubated at 37 °C for 2 h, blocked with 5% BSA in PBS 0.1% Tween-20, and incubated at 37 °C for 1 h. The rPvAARP-FL-coated slides were reacted with sera from malaria patients or healthy individuals (1:25 dilution) at 37 °C for 1 h. Alexa Fluor 546-conjugated goat anti-human IgG (10 µg/mL; Invitrogen Life Technologies) was used as a secondary antibody, and the fluorescence signal was detected with a scanner (InnoScan scanner, Carbonne, France). The cut-off values were set to the mean fluorescence intensity (MFI) plus 2 standard deviations (SDs). The normalized MFI was calculated by dividing the MFI by the cut-off value.

Amine-coated slides were used and spotted in duplicate with 100 ng of purified recombinant proteins of PkDBPα or PkAMA1 at 37 °C for 2 h. After blocking with 5% BSA in PBS 0.1% Tween-20, knowlesi-infected patients’ serum and healthy individuals’ serum were reacted (1:25) for 1 h. Alexa Fluor 546-conjugated goat anti-human IgG (10 µg/mL; Invitrogen Life Technologies) was used as a secondary antibody, and the fluorescence signal was detected with a scanner (InnoScan scanner, Carbonne, France). The cut-off values were set to the mean fluorescence intensity (MFI) plus 2 standard deviations (SDs). The normalized MFI was calculated by dividing the MFI by the cut-off value.