Antigens were coupled to MagPlex-C microspheres (Luminex, Austin, TX) as described previously, with 1 million microspheres coupled with 1 µg of MSP-1, AMA-1, and TTc and 15 µg of CSP [36 (link)]. Immunoglobulin G was measured in a multianalyte platform assay as previously described [30 (link), 36 (link)]. In brief, 50 µL (2000 microspheres/test) antigen-coupled microspheres were incubated with 50 µL maternal and cord plasma at 1:100 dilution in phosphate-buffered saline (PBS) and 1% bovine serum albumin (BSA) for 1 hour at 25°C on a shaker. After 3 washes, microspheres were incubated with 100 µL secondary antibody diluted to 2 µg/mL in PBS-1% BSA per well (R-phycoerythrin-conjugated, Affini Pure F(ab′)2 fragment, goat anti-human IgG Fc fragment specific; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour. Microspheres were washed 3 times, resuspended in 100 µL PBS-1% BSA, and analyzed using a MAGPIX reader (EMD Millipore, Billerica, MA). The results were expressed as median fluorescence intensity (MFI). Positive and negative controls were included on each plate consisting of (1) pool of plasma from 6 Cameroonian adults (positive assay control) and (2) pool of plasma from 13 North Americans (negative assay control). In addition, plasma from 20 North American adults was used to determine the cutoff for antibody positivity.