Detection of a Plasmodium vivax erythrocyte binding protein by flow cytometry

被引:32
作者
Tran, TM
Moreno, A
Yazdani, SS
Chitnis, CE
Barnwell, JW
Galinski, MR
机构
[1] Emory Univ, Emory Vaccine Ctr, Yerkes Natl Primate Res Ctr, Atlanta, GA 30329 USA
[2] Int Ctr Genet Engn & Biotechnol, Malaria Res Grp, New Delhi, India
[3] Ctr Dis Control & Prevent, Natl Ctr Infect Dis, Div Parasit Dis, Malaria Branch, Atlanta, GA USA
[4] Emory Univ, Dept Med, Div Infect Dis, Atlanta, GA 30322 USA
关键词
malaria; flow cytometry; Plasmodium vivax; reticulocyte; erythrocyte binding assay; Duffy binding protein;
D O I
10.1002/cyto.a.20098
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 (T-EBAs) that has several advantages over traditional erythrocyte binding assays (F-EBA) 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 isolate 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. (C) 2004 Wiley-Liss, Inc.
引用
收藏
页码:59 / 66
页数:8
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