Figure 4

Detection of native Pf MSP4 by recombinant 2.44IgG1. (A) Specific binding of the murine 2.44IgG1 to methanol-fixed Pf 3D7A schizonts was assessed by IFA. The murine 2.44IgG1 was visualized with AlexaFluor®488-conjugated goat anti-mouse IgG secondary antibody (green) and AMA1 on the parasite surface was stained with Ra-AMA1 which was visualized with AlexaFluor®594 conjugated secondary antibody (red), showing the circular localization of AMA1 in mature schizonts. Nuclei were stained with Hoechst 33342 (blue). The overlay image includes the bright field image. The small overlay shows the lack of green staining obtained with an irrelevant murine IgG. Scale bar = 2.5 μm. (B) AMA1 on the parasite surface was stained with Ra-AMA1 and visualized with AlexaFluor®488-conjugated secondary antibody (green), showing a circular localization of AMA1 indicating fully mature schizonts. The recombinant 2.44IgG1 was visualized with a Cy3-conjugated goat anti-human IgG secondary antibody (red). Nuclei were stained with Hoechst 33342 (blue). The overlay image includes the bright field image. The small overlay shows the lack of red staining obtained with an irrelevant chimeric IgG. Scale bar = 2.5 μm. (C) Detection of PfMSP4 by recombinant 2.44IgG1 using western blot analysis. A western blot of SDS-PAGE-separated merozoite extract was probed with 4 μg of the recombinant 2.44IgG1 revealing one specific band (~40 kDa), indicated with an arrow, which corresponds to the anticipated molecular size of PfMSP4.