Optiplate microtiter plates

Interaction between PfMSA180 and 13 erythrocyte surface proteins was quantified by AlphaScreen as reported with slight modification^{35 (link),36 (link)} (Fig. S2A). Briefly, reactions were carried out in 20 μl of reaction volumes per well in 384-well OptiPlate microtiter plates (PerkinElmer). Affinity-purified PfMSA180-Tr4 recombinant protein was biotinylated using a Biotin Labeling Kit-NH₂ (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s instructions. Then 5 μl of 10 nM biotinylated protein was mixed with 5 μl of 10 nM of each erythrocyte surface protein in reaction buffer (100 mM Tris-HCL [pH 8.0], 0.01% [v/v] Tween-20 and 0.1 mg/ml [w/v] bovine serum albumin) and incubated for 1 h at 26 °C to form a protein-protein complex. Subsequently, a 10 μl suspension of streptavidin-coated donor-bead and anti-GST acceptor-bead (PerkinElmer) 1:1 (v/v) mixture in the reaction buffer was added to a final concentration of 15 μg/ml of both beads. The mixture was incubated at 26 °C for 12 h in the dark to allow the donor- and acceptor-beads to optimally bind to biotin and GST, respectively. Upon illumination of this complex, a luminescence signal at 620 nm was detected using an EnVision plate reader (PerkinElmer) and the result was expressed as AlphaScreen counts. HisGST was used to set the background and was subtracted from each sample signal.

Interaction between PfRipr and 13 erythrocyte surface proteins was quantified by AlphaScreen as reported^{40 (link)}. Briefly, reactions were carried out in 20 µl of reaction volume per well in 384-well OptiPlate microtiter plates (PerkinElmer). First, affinity-purified Ecto-PfRipr recombinant protein was biotinylated using a Biotin Labeling Kit-NH₂ (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s instruction. Secondly, 5 µl of 10 nM biotinylated protein was mixed with 5 µl of 10 nM for each erythrocyte surface protein in reaction buffer (100 mM Tris-HCL [pH 8.0], 0.01% [v/v] Tween-20 and 0.1 mg/ml [w/v] bovine serum albumin), and incubated for 1 h at 26 °C to form a protein-protein complex. Subsequently, a 10 µl suspension of streptavidin-coated donor-beads and anti-GST acceptor-beads (PerkinElmer) mixture in 1:1 (v/v) in the reaction buffer was added to a final concentration of 15 µg/ml of both beads. The mixture was incubated at 26 °C for 12 h in the dark to allow the donor- and acceptor-beads to optimally bind to biotin and GST, respectively. Upon illumination of this complex, a luminescence signal at 620 nm was detected by the EnVision plate reader (PerkinElmer) and the results were expressed as AlphaScreen counts. GST tagged Rh5, known to interact with PfRipr, was included as a positive control and His-GST as a negative control.

Amplified luminescence proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) was employed to measure the serum anti-CSF2 antibody (s-CSF2-Ab) and serum anti-bCSF2-77-peptide antibody (s-CSF2pep-Ab) levels. The reaction mixture containing 2.5 μL of serum samples diluted at 1:100 in AlphaLISA buffer (25 mM HEPES pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL of dextran-500, and 0.05% Proclin-300) and 2.5 μL of GST or GST-CSF2 proteins (10 μg/ml) or 400 ng/mL of bCSF2-77 was incubated in 384-well white opaque OptiPlate microtiter plates (PerkinElmer, Beaconsfield, United Kingdom) at room temperature for 6–8 h. Next, 2.5 μL of anti-human immunoglobulin G (IgG)-conjugated acceptor beads (40 μg/ml) and 2.5-μL glutathione-conjugated donor beads (40 μg/ml) or 2.5 μL of streptavidin-conjugated donor beads (40 μg/ml) were added. After incubation in the dark for 7–28 days at room temperature, chemical emission was measured using an EnSpire Alpha microplate reader (PerkinElmer), as previously described (48 (link), 51 (link), 52 (link)). Specific reactions were calculated by subtracting the emitted alpha photon counts of the GST and buffer controls from those of the GST-CSF2 protein and the bCSF2-77 peptide, respectively.