Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites.
BACKGROUND
Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites.


METHODS
After surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels.


RESULTS
Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in surface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3.


CONCLUSIONS
Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations.
Sharling
Lisa
L
Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, King's Buildings, Edinburgh, Scotland, UK. sharling@uga.edu <sharling@uga.edu>
Sowa
Kordai Mp
KM
Thompson
Joanne
J
Kyriacou
Helen M
HM
Arnot
David E
DE
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis / Q000737:chemistry*; D001710:Biotin / Q000032:analysis* / Q000737:chemistry*; D019921:Biotinylation / Q000379:methods*; D002478:Cells, Cultured; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology; D016788:Plasmodium chabaudi / Q000737:chemistry* / Q000378:metabolism; D010963:Plasmodium falciparum / Q000737:chemistry* / Q000378:metabolism
D000954:Antigens, Surface; D001710:Biotin