Three experiments. (F ) Determination on the specificity of mAb F8A1.1 by ELISA employing defined schistosome antigen (LDNF epitope), S. mansoni SEA and glycoproteins. (F) Wells had been coated with LDNFPBSA, SEA, KLH, HRP or PLA2 and incubated with F8A1.1 and detected with antimouse IgG. (G) Crossreactivity of antigens in (F) against 1 : one hundred dilution of 10 weeks sera from mice infected with S. mansoni. (H) The presence of Fuc in antigens. Wells have been coated as in (F) and incubated with biotinylated AAL as in (E). Error bars represent means 1 SD from three replicate readings within a single experiment; representative of three experiments.M Mandalasi et al.of SFM. The purity from the purified F8A1.1 was determined by sodium dodecyl sulfate olyacrylamide gel electrophoresis (SDS AGE) analysis below minimizing situations and Coomassie blue staining, using total mouse IgG as a handle. Two protein bands representing heavy and light chain subunits were obtained for each the purified F8A1.1 and also the manage IgG indicating that a higher degree of purity of F8A1.1 was obtained (Figure 1B). The isotype in the purified F8A1.1 was determined by enzymelinked immunosorbent assay (ELISA) using SEA and LNFPIIIBSA as antigenic targets and antimouse IgM, IgG, IgG1, IgG2a, IgG2b or IgG3 for detection of bound antibodies. F8A1.1 bound to both SEA and LNFPIIIBSA and was detectable by both antimouse IgG and IgG3, but not by antimouse IgM, IgG1, IgG2a or IgG2b (Figure 1C), indicating that F8A1.1 is an IgG3 that is potentially able to recognize Lex epitopes. We further tested the binding of F8A1.1 in ELISA toward a panel of neoglycoproteins expressing a variety of different fucosylated glycan antigens (Nyame et al. 2000). The neoglycoproteins used incorporated lactoNfucopentaose IIBSA (LNFPIIBSA; Gal13(Fuc14)GlcNAc13Gal14GlcBSA), which bears the Lewis a trisaccharide epitope; lactoNdifucohexaose IBSA (LNDFHIBSA; Fuc12Gal13 (Fuc14)GlcNAc13Gal14GlcBSA), which expresses the Lewis b tetrasaccharide epitope; lactoNfucopentaose IBSA (LNFPIBSA; Fuc12Gal13Gal14GlcBSA), which expresses blood group H (form I) epitope; and lactoNneotetraoseBSA (LNnTBSA; Gal14GlcNAc13Gal14GlcBSA), which bears the LN glycan backbone.888725-91-5 site F8A1.3-Ethynyltetrahydrofuran Chemical name 1 bound to LNFPIIIBSA but to not other fucosylated glycan antigens tested (Figure 1D).PMID:25269910 To rule out the possibility that the observed outcome could possibly be on account of variations in antigen coating efficiencies, neoglycoconjugate coated wells had been incubated with biotinylated Aleuria aurantia lectin (AAL) and probed with peroxidase conjugated streptavidin to estimate the densities in the coated neoglycoproteins by the density of their Fuc residues (Kochibe and Furukawa 1980). AAL bound to all the fucosylated neoglycoproteins with equivalent intensities and also the binding was totally inhibited with cost-free Fuc, hence confirming the specificity on the AAL binding. This result indicates that the observed binding of F8A1.1 to LNFPIIIBSA was not as a result of differences in antigen coating efficiency (Figure 1E) but due to the apparent specificity of the F8A1.1 for the Lex epitope. To additional characterize the specificity of F8A1.1, we tested its binding in ELISA against a number of antigenic targets that happen to be bound by antibodies in sera of schistosome infected humans and animals. The antigens analyzed incorporated lacdiNAc fucopentaoseBSA (LDNFPBSA; GalNAc14 (Fuc13)GlcNAc13Gal14GlcBSA), which bears the defined fucosylated lacdiNAc (LDNF; GalNAc14(Fuc13) GlcNAcR) antigens of schistosomes, horseradish peroxid.
