Fluorescently labeled liposomes for monitoring cholera toxin binding to epithelial cells.
Vibrio cholerae, the causative agent for cholera, expresses a toxin required for virulence consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB is frequently used as an indicator of the presence of pathogenic V. cholerae and binds to the G(M1) ganglioside on the surface of epithelial cells. To study V. cholerae virulence (CTB expression) in the presence of human epithelia, we devised an inexpensive, simple, and rapid method for quantifying CTB bound on epithelial surfaces in microtiter plates. G(M1) ganglioside was incorporated into the lipid bilayer of liposomes both encapsulating the fluorescent dye sulforhodamine B (SRB) and with SRB tagged to lipids in the bilayer (BEGs). In addition, G(M1)-embedded liposomes encapsulating SRB only (EGs) and with SRB in their bilayers only (BGs) were synthesized. The three types of liposomes were compared with respect to their efficacy for both visualizing and quantifying CTB attached to the surface of Caco-2 cells. The BEGs were the most effective overall, providing both visualization under a fluorescence microscope and quantification after lysis in a microtiter plate reader. A limit of detection corresponding to 0.2 8microg/ml applied CTB was attained for the on-cell assay using the microtiter plate reader approach, whereas as low as 2 microg/ml applied CTB could be observed under the fluorescence microscope.
Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA.
Katie A
KA
Edwards
Faping
F
Duan
Antje J
AJ
Baeumner
John C
JC
March
D018938:Caco-2 Cells; D049109:Cell Proliferation; D002772:Cholera Toxin / Q000032:analysis* / Q000378:metabolism*; D018920:Coculture Techniques; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D015203:Reproducibility of Results; D012235:Rhodamines / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013501:Surface-Active Agents / Q000494:pharmacology; D014734:Vibrio cholerae / Q000235:genetics
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D012235:Rhodamines; D013501:Surface-Active Agents; C022027:lissamine rhodamine B; D002772:Cholera Toxin