Production and characterization of monoclonal antibodies specific to lactotriaosylceramide (original) (raw)
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Archives of Biochemistry and Biophysics, 1997
also shown to detect the accumulated oligosaccharides with nonreducing terminal b-GlcNAc residues as gran-We generated four monoclonal antibodies (MAbs) ular inclusions in the cultured fibroblasts from a classpecific for asparagine-linked neutral oligosacchasical Sandhoff disease patient. ᭧ 1997 Academic Press rides of glycoproteins by immunizing mice with neo-Key Words: monoclonal antibody; N-linked oligosacglycolipids, which were derived from glycoproteins by charide; neoglycolipid. conjugation to phosphatidylethanolamine dipalmitoyl. The binding specificity of these MAbs was determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography.
Carbohydrate Research, 1983
AHSTRACT Two mouse hybridoma antibodies (LICR-LON-M39 and LICR-LON-M18) against the human-milk-fat globules were found to resemble human autoantibodies of anti-1 type in their cold aggiutinating property and their preferential reactions with erythrocytes of I-rather than i-type. From inhibition of binding assays with glycoproteins having known A. B. H, Le". Le ', I, and i activities, and oligosaccharides of the Type I and Type 2 lacto-ltl-glycosyl series, it was established that these antibodies are directed at Type 2 structures, and that the I(Ma) determinant, P-D-Galp-(l-+4)-/3-D-GlcpNAc-(l-+6), which is usually found on branched oligosaccharides. is the preferred sequence. The hybridoma antibodies as well as anti-1 Ma were shown to react well with the P-D-Galp-(l-+4)-p-D-GlcpNAc-(l-+6)-D-Gal or-D-Man sequence. Studies of the reactions of these antibodies with glycolipids on thin-layer plates showed that the two hybridoma antibodies differ from anti-I Ma in reacting weakly with the unbranched i-type sequence p-D-Galp-(1~4)-~-D-GIcpNAc-(l~3)-P_D-Gal~-(l~4)-~-D-Glc~NAc-{l~3)-~-D-Gal~-(l-+4) as found on ~~c~u-~-~~~hexaosylceramide. Furthermore, they differ from anti-I Ma but resemble anti-1 Woj and Sti, and a hybridoma antibody lB2 in their failure to react with their determinant in the presence of aD -(l-+3)-linked galactosyl groups. From their lack of reactions with blood-group-A and-H active glycoproteins, and their reactions with neuraminidase-treated erythrocytes, it was deduced that the determinants recognised by the two hybridoma antibodies are also masked in the presence of cu-L-(l-+2)-linked fucosyl groups and sialic acid.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1985
Six monoclonal antibodies with known specificities for the carbohydrate antigens i, X or Y, and seven anti-myeloid antibodies (determinants unknown) selected for their differing reaction patterns with human leucocytes were tested in chromatogram binding assays for reactions with myeloid cell glycolipids derived from normal human granulocytes and chronic myelogenous leukemia cells. Antigenicities were found exclusively on minor glycolipids which were barely or not at all detectable with orcinol-sulphuric acid stain. Among these, a neutral glycusphingolipid bound the anti-i antibody Den and chromatographed as the ceramide octasaccharide, Gait1-, 4GicNAc~I ~ 3Gait1 ~ 4GIcNAcfll ~ 3Gait1 ~ 4GlcNAc~1-, 3Gait1-> 4GIc-Cer. Several species of neutral glycosphingolipids with six to more than ten monosaccharides were detected which carry the X antigen and others the Y antigen: Galfll-, 4(Fucal ~ 3)GIcNAc and Fucal ~ 2Galfll-*4(Fucal ~3)GIcNAc, respectively. In addition, three new types of carbohydrate specificities were detected among the myeloid cell glycolipids. Two were associated with neutral glycolipids: the first, recognised by anti-myeloid antibodies VIM-1 and VIM-10, was expressed on a distinct set of glycolipids with six or more monosaccharides, and the second, recognized by VIM-8, was expressed on glycolipids with more than ten monosaccharides. The third specificity, recognised by the anti-myeloid antibody VIM-2, was expressed on slow migrating sialoglycolipids with backbone structures of the poly-Nacetyilactosamine type that are susceptible to degradation with endo-fl-galactosidase. Thus, we conclude that the i and Y antigens occur among the glycolipids of normal myeloid and chronic myelogenous leukemia cells and that a high proportion of hybridoma antibodies raised against differentiation antigens of myeloid cells are directed at carbohydrate structures.
International Journal of Cancer, 1984
Two monoclonal antibodies (KH-I and KH-2) against a transplanted fibrosarcoma (KMT-17) in WKA rab were produced by fusing a mouse myeloma (Pl-X63-Ag8.653) with spleen cells from syngeneic rats hyperimmunized with KMT-17. Both antibodies showed complement-dependent cytotoxicity against KMT-17. By absorption of cytotoxicity. KH-I reacted with homologous tumor, other syngeneic fibrosarcomas (KMT-80 and KMT-75). and lung and kidney from normal rats. However, KH-2 reacted with many kinds of tumors and various normal tissues. Antigen specificity was tested by complement fixation and/or solidphase radioimmunoassay using glycolipids isolated from KMT-17 cells and authentic glycolipids. KH-I reacted with globotriglycosyl ceramide which war not detected on
Glycobiology, 2012
Glycosphingolipids (GSLs) are information-bearing biomolecules that play critical roles in embryonic development, signal transduction and carcinogenesis. Previous studies indicate that certain GSLs are associated with differentiation in acute myeloid leukemia (AML) cells. In this study, we collected bone marrow samples from healthy donors and AML patients and analyzed the GSL expression profiles comprehensively using electrospray ionization linear ion-trap mass spectrometry. The results showed that AML patients had higher expression of the GSL lactotriaosylceramide (Lc3), GM3 and neolactotetraosylceramide (nLc4) in their bone marrow than did the healthy donors (P < 0.05), especially the M1 subtype of AML. To further explore the molecular mechanisms of Lc3, we examined the expression of the Lc3 synthase β1,3-N-acet-ylglucosaminyltransferase5 (β3Gn-T5) and found that the bone marrow samples of AML patients had 16-fold higher expression of β3Gn-T5 than those of healthy donors (P < 0.05). Our results suggest that AML-associated GSLs Lc3, GM3 and nLc4 are possibly involved in initiation and differentiation of AML.