Detection of a Plasmodium vivax erythrocyte binding protein by flow cytometry

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Abstract
Background The malaria parasite Plasmodium vivax preferentially invades reticulocytes. It is therefore relevant for vaccine development purposes to identify and characterize P. vivax proteins that bind specifically to the surface of reticulocytes. We have developed a two‐color flow cytometric erythrocyte binding assay (F‐EBA) that has several advantages over traditional erythrocyte binding assays (T‐EBAs) used in malaria research. We demonstrate the use of F‐EBA using the P. vivax Duffy binding protein region II (PvDBP‐RII) recombinant protein as a model. This protein binds to all erythrocytes that express the Duffy receptor (Fy) and discriminates binding between normocytes and reticulocytes. Methods F‐EBAs were performed by incubating freshly isolated Aotus nancymai, Macaca mulatta, Saimiri boliviensis, and human erythrocytes with PvDBP‐RII, a fluorescent anti‐His tag detection antibody, and thiazole orange before flow cytometric analysis. T‐EBAs employing immunoblot detection with an anti‐His antibody were performed concomitantly. Results PvDBP‐RII bound to A. nancymai, M. mulatta, and human Fy+ erythrocytes, but not human Fy− erythrocytes, by F‐EBAs and T‐EBAs. However, F‐EBAs exhibited higher sensitivity and better concordance between experiments compared with T‐EBAs. Conclusions F‐EBA is a rapid, simple, and reliable method for quantifying the ability of malaria proteins to bind to the surface of erythrocytes. F‐EBA can discriminate binding between erythrocyte subpopulations without enrichment protocols and may be more reliable and sensitive than T‐EBAs in identifying novel erythrocyte binding proteins.