Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry

Background: Naturally induced antibodies binding to surface antigens of Pla smodium falciparum-infected erythrocytes can be detected by direct agglutin ation of infected erythrocytes or by indirect immunofluorescence on intact, unfixed, infected erythrocytes. Agglutinating antibodies have previously b een shown to recognise Plasmodium falciparum erythrocyte membrane protein I (PfEMP1). This protein is inserted by the parasite into the host cell memb rane and mediates the adhesion to the venular endothelium of the host organ ism in vivo. Methods: Erythrocytes infected at high parasitaemias with ethidium-bromide- labelled mature forms of P.falciparum parasites were sequentially exposed t o immune plasma, goat anti-human immunoglobulin (Ig) G, and fluorescein-iso thiocyanate-conjugated rabbit anti-goat Ig. Plasma antibodies recognising a ntigens exposed on the surface of parasitised erythrocytes were subsequentl y detected by two-colour flow cytometry. Results: Binding of human antibodies to the surface of erythrocytes infecte d with adhesive strains of Plasmodium falciparum can be measured by the two -colour flow cytometry (FCM) assay described. In addition, we demonstrate t hat the adhesive capacity of a parasite isolate correlates with the capacit y of human immune plasmas to label the isolate as detected by FCM. We also show that the antigens recognised by the labelling antibodies are strain sp ecific and that their molecular weights are in the range previously describ ed for PfEMP1 antigens. Conclusions: Our FCM assay predominantly detects antibodies that recognise PfEMP1 and thus constitutes a convenient assay for the analysis of acquisit ion, maintenance, and diversity of anti-PfEMP1-specific antibodies and for the examination of class and subclass characteristics. (C) 1999 Wiley-Liss, Inc.