Development and characterization of a specific IgG monoclonal antibody toward the Lewis x antigen using splenocytes of Schistosoma mansoni-infected mice

摘要

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.