Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites

<p>Abstract</p> <p>Background</p> <p>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 <it>ex-vivo </it>parasites.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled <it>Plasmodium falciparum </it>and <it>Plasmodium chabaudi </it>erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of <it>P. falciparum-infected </it>erythrocytes is notably enriched in suface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3.</p> <p>Conclusion</p> <p>Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both <it>in vitro </it>and <it>ex vivo Plasmodium </it>parasite preparations.</p

A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in Plasmodium falciparum infected erythrocytes

<p>Abstract</p> <p>Background</p> <p>The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of <it>Plasmodium falciparum </it>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.</p> <p>Methods</p> <p>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</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in infected erythrocytes-5

Asitized cell. 3D7 infected erythrocytes are shown at three different stages of development with different amounts of surface labelled Var4 PfEMP1 (green Alexa488 fluorescence). Red Alexa568 fluorescence indicates intracellular reaction with the anti-PfEMP1 antisera. Nuclei are stained with DAPI. Scale bar 5 μM.<p><b>Copyright information:</b></p><p>Taken from "A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in infected erythrocytes"</p><p>http://www.malariajournal.com/content/7/1/101</p><p>Malaria Journal 2008;7():101-101.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2453135.</p><p></p

Summary of the validation experiments demonstrating specific labelling and distribution of surface and internal PfEMP1 antigens in infected erythrocytes

<p><b>Copyright information:</b></p><p>Taken from "A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in infected erythrocytes"</p><p>http://www.malariajournal.com/content/7/1/101</p><p>Malaria Journal 2008;7():101-101.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2453135.</p><p></p

A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in infected erythrocytes-4

Cted erythrocyte, surface-labelled with fluorescent secondary antibodies recognising the anti-VAR2CSA antibodies, in the presence of the membrane permeable nuclear dye ethidium bromide.<p><b>Copyright information:</b></p><p>Taken from "A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in infected erythrocytes"</p><p>http://www.malariajournal.com/content/7/1/101</p><p>Malaria Journal 2008;7():101-101.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2453135.</p><p></p

A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in infected erythrocytes-0

Rimposed. All even numbered plates show the fluorescence image alone. The series shows antibody binding to unselected FCR3 infected erythrocytes. Plates in the series show FCR3 infected erythrocytes which have been pre-selected for adhesion to BeWo cells. Before preparation for microscopy magnetically purified cells were seeded with control uninfected erythrocytes. The antibodies used in each row of images are indicated on the left. Scale bar 5 μM.<p><b>Copyright information:</b></p><p>Taken from "A method for visualizing surface-exposed and internal PfEMP1 adhesion antigens in infected erythrocytes"</p><p>http://www.malariajournal.com/content/7/1/101</p><p>Malaria Journal 2008;7():101-101.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2453135.</p><p></p