IgG reactivity against recombinant proteins was measured by means of enzyme-linked immunosorbent assay (ELISA) as described elsewhere [20 (link)]. Briefly, plasma samples (1:400) followed by horseradish peroxidase–conjugated rabbit anti-human IgG (1:3000; Dako) were added to 96-well flat-bottom microtiter plates (Nunc MaxiSorp; Thermo Fisher Scientific) previously coated with recombinant proteins. Bound antibodies were detected by adding TMB PLUS2 (Eco-Tek), and the reaction stopped with 0.2 mol/L sulfuric acid. The optical density was read at 450 nm (HiPo MPP-96 microplate reader; Molecular Devices), and the specific antibody levels were calculated in arbitrary units, as described elsewhere [20 (link)]. Plasma samples from Danish adults without malaria exposure and a pool of Ghanaian adults with previous P. falciparum infection were included as negative and positive controls, respectively. Negative cutoff values were calculated as the mean arbitrary unit values plus 2 standard deviations obtained with the negative control samples.
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