title: Comparison of detection methods for cell surface globotriaosylceramide.
abstract: The cell surface-expressed glycosphingolipid (GSL), globotriaosylceramide (Gb(3)), is becoming increasingly important and is widely studied in the areas of verotoxin (VT)-mediated cytotoxicity, human immunodeficiency virus (HIV) infection, immunology and cancer. However, despite its diverse roles and implications, an optimized detection method for cell surface Gb(3) has not been determined. GSLs are differentially organized in the plasma membrane which can affect their availability for protein binding. To examine various detection methods for cell surface Gb(3), we compared four reagents for use in flow cytometry analysis. A natural ligand (VT1B) and three different monoclonal antibodies (mAbs) were optimized and tested on various human cell lines for Gb(3) detection. A differential detection pattern of cell surface Gb(3) expression, which was influenced by the choice of reagent, was observed. Two mAb were found to be suboptimal. However, two other methods were found to be useful as defined by their high percentage of positivity and mean fluorescence intensity (MFI) values. Rat IgM anti-Gb(3) mAb (clone 38-13) using phycoerythrin-conjugated secondary antibody was found to be the most specific detection method while the use of VT1B conjugated to Alexa488 fluorochrome was found to be the most sensitive; showing a rare crossreactivity only when Gb(4) expression was highly elevated. The findings of this study demonstrate the variability in detection of Gb(3) depending on the reagent and cell target used and emphasize the importance of selecting an optimal methodology in studies for the detection of cell surface expression of Gb(3).
authors: Institute of Medical Science, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8.
authors: Minji
authors: M
authors: Kim
authors: Beth
authors: B
authors: Binnington
authors: Darinka
authors: D
authors: Sakac
authors: Kimberly R
authors: KR
authors: Fernandes
authors: Sheryl P
authors: SP
authors: Shi
authors: Clifford A
authors: CA
authors: Lingwood
authors: Donald R
authors: DR
authors: Branch
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry; D002855:Chromatography, Thin Layer; D005434:Flow Cytometry / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007202:Indicators and Reagents; D019169:Jurkat Cells; D008024:Ligands; D008563:Membrane Lipids / Q000032:analysis / Q000276:immunology; D051381:Rats; D022622:Shiga Toxin 1; D014281:Trihexosylceramides / Q000032:analysis* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D008024:Ligands; D008563:Membrane Lipids; D022622:Shiga Toxin 1; D014281:Trihexosylceramides; C018549:globotriaosylceramide