Integrin α5β1

Ring stage parasite samples were allowed to mature to the trophozoite/schizont stages during in vitro culture for 16–24 h, then mature parasite forms were enriched by gelatin flotation. Parasite binding inhibition assay was performed using a parasite suspension at 2–20% parasitaemia and 0.5% haematocrit [29 (link)]. 20 µl of recombinant endothelial receptors (CD36, ICAM-1, PECAM-1, P-selectin, integrin α₃β₁, integrin α₅β₁, integrin α_vβ₃, JAM-B, from R&D Systems Minneapolis, MN, cellular fibronectin, and laminin from placenta from Sigma, St. Louis, MO) at a concentration of 10 µg/ml were immobilized by adsorption on Petri dishes. The parasite suspension was preincubated with plasma diluted 1:5 for 30 min at room temperature then allowed to bind to the immobilized receptor for 30 min at room temperature. Plates were washed three times with PBS to remove unbound cells. Bound cells were fixed in 0.5% glutaraldehyde for 10 min, stained with 10% Giemsa for 2 min and quantified by counting the number of parasites in 20 high-power fields. Level of inhibition was defined based on the level of IE binding in the presence of a pool of plasma samples collected from malaria-naïve adults in the USA (negative control) as follow (100 − (N_test/N_control) × 100)). A minimum IE count of 20 in the presence of naïve plasma was required for the results to be accepted.