Direct Membrane-Feeding Assay (DMFA)

The TB activity of 143 plasma samples (1:2 dilution), and affinity-purified CHO-rPvs48/45 specific IgG antibodies at 10, 40, and 100 μg/mL was measured by a DMFA using P. vivax gametocyte-infected blood obtained from malaria patients, as previously described (9). Briefly, P. vivax infected blood was centrifuged, and cell-pellets containing infected RBC (iRBC) were subsequently reconstituted with a pool of heat-inactivated male AB+ sera obtained from healthy donors purchased from Sigma (H5667 Sigma-Aldrich Inc) or with test samples. A total of ~100 Anopheles albimanus mosquitoes previously subjected to overnight fasting were fed for 15-20 minutes/cage with this mixture. Mosquito midguts were stained with 2% mercurochrome on day 7 post-feeding, and the number of oocysts per mosquito midgut counted.