Abstract
BackgroundPlasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing an automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin.MethodsChinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO cells, was quantified by microscopy of Giemsa-stained chamber slides. In the automated assay, binding was quantified spectrophotometrically in microtiter plates after cell lysis using tetramethylbenzidine as peroxidase-catalysed substrate. The relevance of the method for binding studies was assessed using: i) binding of P. falciparum-infected erythrocytes to CHO cells over-expressing chondroitin sulfate A and ii) CHO cells transfected with CD36. Binding of infected erythrocytes including field isolates to primary endothelial cells was also performed. Data was analysed using linear regression and Bland-Altman plots.ResultsThe manual and automated quantification showed strong, positive correlation (r2 = 0.959, p <0.001) and with similar detection limit and precision. The automated assay showed the expected dose-dependent reduction in binding to CHO cells when blocking with soluble chondroitin sulfate A or anti-CD36 antibody. Quantification of binding to endothelial cells showed clear distinction between selected vs. non-selected parasite lines. Importantly, the assay was sufficiently sensitive to detect adhesion of field isolates to endothelial cells.ConclusionsThe assay is simple and in a reproducible manner quantifies erythrocyte adhesion to several types of immobilized cells.
Highlights
Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature
Erythrocytes have peroxidase activity and this activity is maintained upon Plasmodium falciparum infection Haem in erythrocytes can act as a pseudoperoxidase [12] and chromogenic substrates have previously been used to quantify haem [13]
When comparing the signal generated from lysed uninfected erythrocytes with lysates from infected erythrocytes, ring-stage erythrocytes or late-stage erythrocytes no difference could be detected (Figure 1B, p >0.7)
Summary
Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The species Plasmodium falciparum accounts for the majority of fatal malaria infections. A central part of pathogenesis and a major determinant for a complicated infection is the parasites’ ability to adhere to endothelial cells in order to avoid splenic clearance [1,2,3]. The majority of binding assays are static binding assays and quantification of infected erythrocytes bound to adherent cells is performed by using the gold standard, manual counting of Giemsa-stained erythrocytes [7,10]. Other ways to quantify cytoadhesion include the use of radioactive 3H-hypoxanthine incorporation
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