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. 2015 Jul 1;61(8):1244–1252. doi: 10.1093/cid/civ525

Figure 1.

Figure 1.

Binding of native erythrocyte-binding antigen 175 (EBA-175) to erythrocytes and inhibition of binding by human antibodies. The degree and specificity of native EBA-175 binding to the surface of intact human erythrocytes was assessed by flow cytometry and Western blot. A, Native EBA-175 from the 3D7 wild-type parasite supernatant (solid line) and 3D7ΔEBA-175 negative controls (dashed line) were assessed with higher fluorescence (x axis), indicating higher EBA-175 binding. B, Western blots were used to confirm the presence or absence of native EBA-175 from parasite culture supernatants using 3D7 wild-type (lane 1) and 3D7ΔEBA-175 (lane 2), respectively. The ability of EBA-175 to bind to human erythrocytes was then assessed by coincubating the parasite supernatants with human erythrocytes, eluting off bound EBA-175, and detecting the presence of EBA-175 by Western blot using a rabbit anti–EBA-175 polyclonal antibody. Binding of EBA-175 was detected with a 175-kDa band when using 3D7wt supernatant (lane 3) but not with 3D7ΔEBA-175 supernatant (lane 4). A faint spectrin band can be seen at 250 kDa. C, The specificity of EBA-175 binding to erythrocytes was assessed following enzyme treatment of the erythrocytes with neuraminidase, trypsin, or chymotrypsin. These enzymes differentially cleave off sialic acids and glycophorin A. Culture supernatants from 3D7 wild-type (black bars) and 3D7ΔEBA-175 (white bars) parasites were used to detect the mean fluorescence intensity (MFI, y axis) of EBA-175 binding to human erythrocytes using the flow cytometry assay described earlier. Error bars show range for samples tested in duplicate. D, Human sera samples were used to assess the ability to inhibit the binding of EBA-175 to human erythrocytes in the flow cytometry assay described earlier, with binding-inhibitory samples yielding a low MFI (y axis). Representative samples included adults resident in malaria-endemic Papua New Guinea (PNG) (n = 2) and samples from malaria-naive blood donors resident in Melbourne, Australia (Melb.) (n = 2). The line graph shows a titration of sera (x axis) tested singly.