Assessment of the strength of P. falciparum IRBC binding to placental CSPG and bCSA. The strength of IRBC binding to human placental CSPG or bCSA was determined by measuring the dose-dependent inhibition of IRBC binding by 36% C4S. The plastic Petri plates were coated with 200 ng/ml solution of CSPG or 50μg/ml solution of bCSA and blocked with 2% BSA. IRBCs (4% suspension in PBS) were incubated with 2 times the indicated concentrations of partially sulfated C4S and then overlaid onto the coated spots. The unbound cells were washed off and the bound cells were fixed, stained with Giemsa, and counted. Assays were carried out two times each in duplicate and percent binding (means ± standard errors of the means) with respect to control IRBC binding is plotted. A, 3D7-CSPG; B, FCR3-CSPG; C, CS2-CSPG; D, 3D7-bCSA; E, FCR3-bCSA. The concentrations of soluble C4S required for 50% inhibition of IRBC binding was considered as the direct measure of the binding strength of IRBCs. For each parasite studied, the strength of IRBC binding was ~2-fold higher for CSPG than bCSA.