An automated method for determining the cytoadhesion of Plasmodium falciparum-infected erythrocytes to immobilized cells

Malaria Journal

Methodology

Casper Hempel¹  Ida M Boisen²  Jørgen AL Kurtzhals²  Trine Staalsø³  Akinwale Efunshile⁴ 

[1] Centre for Medical Parasitology at Department of Clinical Microbiology, Copenhagen University Hospital Department Clinical Microbiology, 7602, Ole Maaløesvej 26, 2200, Copenhagen N, Denmark;Centre for Medical Parasitology at Department of Clinical Microbiology, Copenhagen University Hospital Department Clinical Microbiology, 7602, Ole Maaløesvej 26, 2200, Copenhagen N, Denmark;Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark;Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark;Institute of Medical Microbiology and Infectious Disease Epidemiology, Medical Faculty, University of Leipzig, Leipzig, Germany;Department of Medical Microbiology, Federal Teaching Hospital/Ebonyi State University, Abakaliki, Nigeria;

关键词: Plasmodium falciparum;    Cytoadhesion;    Chinese hamster ovary cells;    Endothelial cells;    Field isolates;   

DOI  :  10.1186/s12936-015-0632-4

 received in 2014-11-26, accepted in 2015-03-02,  发布年份 2015

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Hempel et al.; licensee BioMed Central. 2015