A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected erythrocytes.
BACKGROUND
The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development.


METHODS
A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy


RESULTS
Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1.


CONCLUSIONS
A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.
Bengtsson
Dominique
D
Centre for Medical Parasitology, Department of International Health, Immunology & Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen K, Denmark. dominique@cmp.dk
Sowa
Kordai M
KM
Salanti
Ali
A
Jensen
Anja Tr
AT
Joergensen
Louise
L
Turner
Louise
L
Theander
Thor G
TG
Arnot
David E
DE
D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion; D004910:Erythrocyte Membrane / Q000469:parasitology / Q000648:ultrastructure; D004912:Erythrocytes / Q000469:parasitology / Q000648:ultrastructure; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000378:metabolism* / Q000472:pathogenicity; D015800:Protozoan Proteins / Q000032:analysis* / Q000737:chemistry; D011817:Rabbits; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods
D015800:Protozoan Proteins; C085731:erythrocyte membrane protein 1, Plasmodium falciparum