title: High yield purification of Plasmodium falciparum merozoites for use in opsonizing antibody assays.
abstract: Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts.  Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies.
authors: Division of Infection and Immunity, Walter and Eliza Hall Institute of Medical Research; Department of Medical Biology, University of Melbourne.
authors: Danika L
authors: DL
authors: Hill
authors: Division of Infection and Immunity, Walter and Eliza Hall Institute of Medical Research; Department of Medical Biology, University of Melbourne.
authors: Emily M
authors: EM
authors: Eriksson
authors: Division of Infection and Immunity, Walter and Eliza Hall Institute of Medical Research; Department of Medical Biology, University of Melbourne; schofield@wehi.edu.au.
authors: Louis
authors: L
authors: Schofield
mesh_terms: D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000276:immunology; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D006420:Hemeproteins / Q000302:isolation & purification; D006801:Humans; D016778:Malaria, Falciparum / Q000276:immunology / Q000469:parasitology; D052940:Merozoites / Q000166:cytology / Q000276:immunology; D009895:Opsonin Proteins / Q000097:blood; D010587:Phagocytosis; D010963:Plasmodium falciparum / Q000166:cytology / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D006420:Hemeproteins; D009895:Opsonin Proteins; C020753:hemozoin