Levels of VAR2CSA-specific IgG were assessed by ELISA as previously described68 (link). In brief, 96-well flat-bottom microtiter plates (Nunc MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4 °C with full-length VAR2CSA (100 ng/well in PBS). Monoclonal antibody (0.08–10 μg/mL) or plasma samples (1:400) were added in duplicate, followed by washing and horseradish peroxidase-conjugated rabbit anti-human IgG (1:3,000; Dako). Bound antibodies were detected by adding TMB PLUS2 (Eco-Tek), and the reaction stopped by the addition of 0.2 M H2SO4. The optical density (OD) was read at 450 nm and the specific antibody levels were calculated in arbitrary units (arb. units), using the equation 100 × [(ODSAMPLE − ODBLANK)/(ODPOS.CTRL − ODBLANK)].
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