The inhibitory capacity of the induced VAR2CSA domainspecific IgG was measured using a standardized Petri dish method both due to the smaller volume in this assay, and because this a more commonly used assay [40] [41] [42] . IgG was purified from the animal sera using HiTrap Protein G HP kit (GE Healthcare) and dialyzed against PBS 16 buffer. Briefly, 20 spots in each Petri dish (Falcon 351005) were coated overnight with 20 ml of decorin (2 mg/ml in PBS). Spots were blocked with 3% bovine serum albumin. Parasite cultures were enriched for late trophozoite and schizont stages in a strong magnetic field (MACS, Miltenyi) and adjusted to reach a 20% parasitemia at 0.5% hematocrit. The parasite suspension was pre-incubated with both animal sera and purified IgG at different concentrations at 37uC for 30 min. Duplicate spots were incubated for 15 min at 37uC in a humid chamber. Unbound erythrocytes were removed by washing with PBS on a gyro-turntable (Stuart) until spots were clear of blood. Bound erythrocytes were glutaraldehyde-fixed (1.5%, 5 min) and Giemsa stained. Five fields were counted in each spot using a magnification of 206 using a Leica Microscope. As negative controls, anti-DBL3cVAR1 serum and IgG were used. Parasite binding to decorin was abrogated by soluble CSA (Sigma-Aldrich) and by chondroitinase (Sigma-Aldrich) treatment of decorin (data not shown).