In vitro growth inhibition assay

In vitro growth inhibition assays with the laboratory-adapted P. falciparum 3D7 clone were performed essentially as described.²⁸ Synchronised trophozoites were adjusted to 0.5% parasitemia and then incubated for 48 h with various concentrations of purified rabbit IgG in PBS at 1% haematocrit. Each culture was set up in triplicate in 96-well flat-bottomed culture plates. Final parasitemia was quantified by flow cytometry after staining of viable parasites with hydroethidine and inhibition calculated relative to infection control wells containing PBS only. The gating strategy used for parasitemia quantification is shown in Supplementary Fig. 3a.

IC₅₀ values for total IgG from PfCyRPA-immunised rabbits were determined in vitro by measuring incorporation of the nucleic acid precursor [³H]-hypoxanthine.²⁹ Infected red blood cells were exposed to increasing concentrations of purified total rabbit IgG in culture plates. After 48 h of incubation, 0.5 μCi [³H]-hypoxanthine was added to each well. Cultures were incubated for further 24 h before they were harvested onto glass-fibre filters and washed with distilled water. The radioactivity was counted using a Betaplate liquid scintillation counter. The results were recorded as counts per minute per well at each IgG concentration and expressed as percentage of the untreated controls. For each individual rabbit a four-parameter sigmoidal dose-response curve was fitted to the relationship between log₁₀(antibody concentration) and % inhibition, and then used to interpolate IC₅₀ values. Data were processed and analysed using GraphPad Prism 7.