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. 2013 Sep 10;8(9):e72504. doi: 10.1371/journal.pone.0072504

Figure 2. Epitopes of highly inhibitory, strain-transcending antibodies are located within the conserved region of EBA-175.

Figure 2

(A) GIA against the homologous 3D7 parasite strain. Rabbits were immunized with 3D7 EBA-175 region III-V. IgG purified from rabbit serum was serially diluted from a starting concentration of 2 mg/ml. Data points indicate mean values of triplicate wells ± standard error of the mean (SEM) in a single assay relative to pre-immune IgG, calculated from a 1-cycle FACS-based assay. The dashed red line indicates the IC50 value of IgG (9 μg/ml). (B) Rabbit IgG against EBA-175 region III-V (3D7) – GIA against homologous and heterologous parasite strains. IgG from a single rabbit were tested against 3D7, W2mef, FCR3 and 3D7Δ175 KO parasite strains. Data points represent mean values of triplicates from 3 independent replicate experiments ± SEM in a 2-cycle assay. The antibody is completely specific to EBA-175 as there is no inhibition of the 175-KO parasites. (C) Rabbit IgG against EBA-175 region III-V (W2mef) – GIA against homologous and heterologous parasite strains. IgG from a single rabbit were tested against 3D7, W2mef, FCR3 and 3D7Δ175 KO parasite strains (2 cycle assay). (D) Anti- EBA-175 RIII-V directly inhibit invasion of purified, viable, D10-GFP merozoites. Results shown are mean + standard error of three independent experiments. (E) Mouse serum against EBA-175 region III-V (3D7) – GIA against homologous and heterologous parasite strains. Three individual mouse sera were pooled and tested against 3D7, W2mef, FCR3 and 3D7Δ175 KO parasite strains (1 cycle assay). (F) GIA patterns of Ig raised against both dimorphic alleles of EBA-175 against a panel of laboratory parasite strains are highly correlated. GIA was performed in a one cycle assay using anti-EBA175RIII-V (3D7) IgG at a concentration of 1 mg/ml. Blue histograms indicate parasite strains that invade predominantly via sialic-acid dependent pathway, red indicates sialic acid-independent pathway [63], grey bars where invasion preference is not known. Parasite strains belonging to the C-type group are shown in blue font whereas those belonging to the F-type are shown in red font. (G) GIA as for (F) but using anti-EBA175RIII-V (W2mef) IgG. (H) GIA using rabbit anti-EBA175 RIV-V (3D7) IgG (One-cycle assay).