Pfspz and iRBCs antibody titers by IFA

NF54 Pfspz, or cultures of iRBCs at 6% parasitaemia were washed twice in PBS/1% BSA. Teflon printed 12-well slides (Electron Microscopy Sciences, Hatfield, PA) were coated with Pfspz (5000/well) or with iRBC (10,000 RBCs/well at 6% parasitaemia) as described [10]. Slides were air dried and stored at − 80 °C until use. Upon thawing, slides were blocked with PBS/1% BSA for 30 min at 37 °C. Twenty microliters of plasma starting at 1:20 and sequential two-fold serial dilutions were added to the wells and incubated for 1 h at 37 °C. Slides were washed three times with PBS, incubated with FITC-labeled goat F(ab′)₂ anti-human IgG (γ chain) or anti-human IgM (µ chain) (Southern Biotech) to measure specific human IgG or IgM antibody titers, or with anti-human IgG (H + L) (BDbiosciences) to measure total specific human antibody titers, for 30 min at 37 °C, washed, and mounted with Vectashield-DAPI (Vector Laboratories).