Characterization of the Duffy-Binding-Like Domain of Plasmodium falciparum Blood-Stage Antigen 332

Figure 5

Detection of native Pf332 in immunofluorescence assays and Western blot. Protein expression of Pf332 in FCR3S1.2 infected red blood cells (iRBC). (a) Immunofluorescence analysis on air-dried monolayers of iRBC (28–32 h p.i.) with human affinity-purified anti-Pf332-DBL antibodies (red) and anti-Pf332-EB200 (green). The parasite was counterstained with DAPI (blue). The antibodies colocalized well and detected Pf332 in Maurer’s cleft like structures in the iRBC cytosol. (b) At 30–34 h p.i., a monoclonal antibody against Pf332-DBL (red) showed colocalization with both anti-Pf332-EB200 (green; upper panel) and anti-MAHRP1, a known Maurer’s cleft marker (green; middle panel). In schizonts, anti-Pf332-DBL (red) could be detected in Maurer’s clefts in close proximity to the RBC plasma membrane. These antibodies did not co-localize with anti-EBA-175 antibodies (green), which gave a punctuate fluorescence pattern typical for antigens localized within the merozoite apical complex. The parasite was counterstained with DAPI (blue). Scale bar indicates 5 μm. (c) Western blot analysis of Pf332 expression in FCR3S1.2 iRBC (36–40 h p.i.). Membranes were probed with human affinity-purified anti-Pf332-DBL (lane 1 and 2), polyclonal anti-Pf332-EB200 (lane 3 and 4), and monoclonal anti-Pf332-DBL (lane 5 and 6). Bands corresponding to full length Pf332 are indicated. All three antibodies recognized Pf332 migrating well above the 170 kDa marker. None of the antibodies reacted with uninfected RBC.