Erythrocytic malaria growth or invasion inhibition assays with emphasis on suspension culture GIA.
Erythrocytic cycle malaria parasite growth or invasion inhibition assays (GIA) compare the effects of various test and control substances on malaria parasite growth in erythrocytes or invasion into erythrocytes in vitro. Although inhibitions by antimalarial drugs in vitro correlate well with drug protective levels required in vivo, as yet there are too few data to know how well inhibitions by antibodies in vitro correlate with the types and degrees of immune protection in vivo. Antibody-mediated GIA is frequently complicated by parasite strain-specific inhibitions, as well as nonspecific inhibitory factors generated in sera collected or stored under nonoptimal conditions. In this chapter, we describe methods for collecting and processing sera, for using different strains of parasite, and a simplified method for staining parasite DNA with Hoechst dye 33342 before quantitating parasites using ultraviolet (UV)-excited flow cytometry. We also describe a new type of GIA using suspension cultures in a 48-well plate. Critical to this method is enclosing the plate in a gassed, heat-sealed plastic bag, which, being low mass, can easily be rested at a 13.5 degrees angle on a rotor platform (114 rpm with 1-in. displacement) to produce gentle pulsatile waves of media in each well. The suspension GIA, which, relative to the static GIA, increased inhibition by one antibody and decreased inhibition by another (Table 1), may better simulate in vivo blood flow and may thus better predict in vivo efficacy.
Department of Immunology, Walter Reed Army Institute of Research, Malaria Program, Naval Medical Research Center, Silver Spring, MD, USA.
J David
JD
Haynes
J Kathleen
JK
Moch
Douglas S
DS
Smoot
D000818:Animals; D004912:Erythrocytes / Q000469:parasitology*; D006801:Humans; D008018:Life Cycle Stages; D008288:Malaria / Q000469:parasitology / Q000503:physiopathology*; D016778:Malaria, Falciparum / Q000469:parasitology / Q000503:physiopathology; D010274:Parasitology / Q000379:methods; D010961:Plasmodium / Q000254:growth & development* / Q000472:pathogenicity; D010963:Plasmodium falciparum / Q000254:growth & development / Q000469:parasitology; D013045:Species Specificity; D014774:Virulence