title: Development and characterization of a specific IgG monoclonal antibody toward the Lewis x antigen using splenocytes of Schistosoma mansoni-infected mice.
abstract: The parasitic blood fluke Schistosoma mansoni synthesizes immunogenic glycans containing the human Lewis x antigen (Le(x); Galactose-β1-4(Fucα1-3)N-acetylglucosamine-β-R, also called CD15), but the biological role(s) of this antigen in the parasites and in humans is poorly understood. To develop IgG-based monoclonal antibodies (mAbs) specific for Le(x), we harvested splenocytes from S. mansoni-infected Swiss Webster mice at Week 10 postinfection, when peak IgG responses to glycan antigens occur, and generated a panel of hybridomas secreting anti-glycan IgG that recognize periodate-sensitive epitopes in soluble egg antigens of the parasites, and also recognizes a neoglycoprotein containing a pentasaccharide with the Le(x) sequence. One murine mAb, an IgG3 designated F8A1.1, bound to glycoproteins and glycolipids from schistosome adults and human promyelocytic leukemic HL-60 cells that express Le(x) antigens, as assessed by a wide variety of approaches including immunofluorescence staining, confocal microscopy, flow cytometry and western blotting, as well as overlay assays of glycolipids after thin-layer chromatography. In contrast, F8A1.1 bound weakly to cercariae, 3-h schistosomula and human Jurkat cells. We also directly compared the glycan specificity of F8A1.1 with commercially available anti-CD15 IgG1 (clone W6D3) using a defined glycan microarray. The results demonstrated that F8A1.1 recognized glycans expressing Le(x) epitopes in a terminal nonreducing position, whereas anti-CD15 bound to glycans with multiple repeats of Le(x) epitopes, but not to glycans with a single, terminal Le(x) epitope. Our results show that F8A1.1 recognizes terminal Le(x) epitopes and can be used for identification, immunolocalization, immunoprecipitation and purification of Le(x)-containing glycoconjugates from schistosomes and mammalian cells.
authors.affiliation: Department of Natural Sciences, University of Maryland Eastern Shore, Princess Anne, MD, USA.
authors.forename: Msano
authors.initials: M
authors.lastname: Mandalasi
authors.forename: Nelum
authors.initials: N
authors.lastname: Dorabawila
authors.forename: David F
authors.initials: DF
authors.lastname: Smith
authors.forename: Jamie
authors.initials: J
authors.lastname: Heimburg-Molinaro
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
authors.forename: A Kwame
authors.initials: AK
authors.lastname: Nyame
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology*; D000918:Antibody Specificity; D000939:Epitopes / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D019169:Jurkat Cells; D016256:Lewis X Antigen / Q000276:immunology*; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D009844:Oligosaccharides / Q000276:immunology; D012550:Schistosoma mansoni / Q000276:immunology*; D012555:Schistosomiasis mansoni / Q000276:immunology*; D013154:Spleen / Q000166:cytology / Q000276:immunology
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D000939:Epitopes; D016256:Lewis X Antigen; D009844:Oligosaccharides
keywords: Lewis x antigen; Schistosoma mansoni; glycans; helminth; monoclonal antibody
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