Victor Ginsburg

Monoclonal antibody A2B5 reacts with many gangliosides in neuronal tissue

Monoclonal antibody A2B5, clone 105, obtained from a mouse immunized with 8-day chicken embryo retinas, reacts with most neuronal cells in chicken retina as revealed by immunofluorescence studies [G.S. Eisenbarth, F.S. Walsh, and M. Nirenberg (1979), Proc. Natl. Acad. Sci. USA 75: 4913-4917]. The antibody binds to many antigens, presumably gangliosides, present in ganglioside fractions from chicken brain, chicken retina, and human brain, as detected by autoradiography of thin-layer chromatograms. Most of the antigens, which are found in the mono-, di-, tri-, and polysialoganglioside fractions, do not correspond in chromatographic mobility to any of the major gangliosides of these tissues, as revealed by orcinol reagent. Apart from the fact that only one neuraminidase-labile sialyl residue is required for binding, the carbohydrate sequence recognized by the antibody is not known. There is a qualitative and quantitative change in the ganglioside antigens in chicken brain and retina during development. The less sialylated antigens appear first, followed by the more sialylated ones. The adult tissues contain little ganglioside antigen.

Many monoclonal antibodies with an apparent specificity for certain lung cancers are directed against a sugar sequence found in lacto-N-fucopentaose III

Abstract Monoclonal antibodies with an apparent specificity for human small-cell carcinoma, adenocarcinoma, and squamous carcinoma of the lung are produced by some hybridomas obtained from mice and rats immunized with an established line of human small cell lung cancer. Out of 85 of these antibodies produced by independently isolated hybridomas from 15 different fusions, 21 are directed against the sugar sequence which occurs in lacto- N -fucopentaose III ceramide, in several higher glycolipids and in glycoproteins. Specificity was determined by autoradiography of thin-layer chromatograms of glycolipids, by solid-phase radioimmunoassays, and by hapten inhibition studies. All 21 antibodies are of the immunoglobulin M type.

The metabolism of L-fucose by HeLa cells.

Abstract HeLa cells incorporate 14 C- l -fucose directly into macromolecules with no detectable randomization of 14 C-activity into other sugars. The pathway of incorporation probably proceeds via GDP- l -fucose, the concentration of which increases markedly when l -fucose is present in the growth medium.

The metabolism of glucosamine by tissue culture cells

Summary The uptake of 14 C-1-glucosamine by HeLa cells and its subsequent fate has been studied. Over 90 per cent of the label initially taken up by the cells is found in the uridine diphosphate- N -acetylhexosamine pool. This pool loses its label in 12 hr and the radioactivity is found in the aminosugar residues of cell components. Eventually, two thirds of the label is secreted into the media as high molecular-weight compounds that contain aminosugar while one third of the label remains with the cells and is gradually metabolized. Labeling of cells in culture with glucosamine 14 C provides a sensitive means for studying cell components that contain aminosugars.

[14] Antibodies against cell surface carbohydrates: Determination of antigen structure

Publisher Summary As the terminal sequences of sugars in glycoproteins and glycolipids are sometimes identical, some antibodies directed against carbohydrates react with both glycoproteins and glycolipids; other antibodies against carbohydrates react only with glycoproteins or only with lycolipids. To obtain cell-specific monoclonal antibodies, mice and rats have been immunized with various cell types in many laboratories. Many of these antibodies are apparently specific for certain cells and developmental stages. This chapter discusses methods (1) for determining whether antibodies that react with cell membranes are directed against carbohydrates; (2) for determining whether the carbohydrate antigens are in glycolipids; (3) glycoproteins, or both; and (4) for determining the structure of the antigens. The chapter presents the steps of analysis of carbohydrate antigens: (1) is total lipid extraction, (2) detection of glycolipid antigens on thin-layer chromatograms by autoradiography, (3) radioimmunoassay of delipidated protein pellet, (4)analysis of monoclonal antibodies that detect glycolipids on thin-layer chromatograms for binding to purified glycolipids using a simple solid-phase radioimmunoassay, (5) immunostained and chemically stained thin-layer chromatograms are compared, (6) enzymatic analysis on TLC, (7) the application of the procedure, DEAE-Sepharose Chromatography, and (8) the chemical analysis of glycolipids, for which the techniques have dramatically changed in the last few years. In some cases, glycolipids are analyzed by enzymatic treatment followed by immunostaining directly on thin-layer chromatograms. Final purification of glycolipids is usually obtained by chromatography on silicic acid.

Leb-active glycolipid in human plasma: Measurement by radioimmunoassay

Abstract A radioimmunoassay that measures Leb-active glycolipids in human plasma has been developed using antiserum from a goat immunized with a Leb blood group hapten, lacto-N-difucohexaose I, conjugated to polylysine. Binding by the antiserum of lacto-N-difucohexaose I conjugated to 125I-labeled bovine serum albumin is specifically inhibited by Leb-active ceramide hexasaccharide. Plasma levels of the glycolipid are quantitated by comparing the inhibitory activity of plasma with that of the purified Leb-active glycolipid. Plasma samples from 35 blood group O Le(a − b +) individuals contain Leb-active ceramide hexasaccharide at an average concentration of 0.9 μg/ml (range: 0.2 to 2.5 μg/ml); no Leb-active glycolipid (less than 0.02 μg/ml) could be detected in plasma from blood group O Le(a + b−) or O Le(a− b−) individuals. Plasma from A1 Le(a − b+) individuals contains less Leb-active glycolipid than plasma from A2 Le(a− b+) individuals: its level in 19 samples of A, Le(a− b+) plasma averages 0.2 μg/ml (range: 0.1 to 0.45 μg/ml), and its level in 9 samples of A2 Le(a− b+) plasma averages 1.1 μg/ml (range 0.8 to 1.3 μg/ml). About one-third of the total Leb-active glycolipid in whole blood is associated with erythrocytes and the rest is found in plasma.

Intracellular site of synthesis of soluble blood group substance

Abstract The intracellular distribution of soluble blood group substance in hog gastric mucosa was investigated. The microsomal fraction contains 1–3% of the total intracellular blood group substance. Labeling experiments in vivo show this small pool to be newly synthesized material and the precursor of other intracellular pools. Attempts to detect a nucleotide-linked, fucose-containing oligosaccharide intermediate in the synthesis of the heterosaccharide chains of blood group substance were unsuccessful.

Glycosphingolipid-binding specificity of the mannose-binding protein from human sera

Mannose-binding protein was purified from human serum to apparent homogeneity by affinity chromatography on mannose-Sepharose, followed by affinity chromatography on underivatized Sepharose. Approximately 0.4 mg protein was obtained from 1 liter serum. The glycosphingolipid-binding specificity of the purified protein was examined by chromatogram overlay and solid phase assays. It binds with high affinity to Lc-3Cer (GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide) and n-Lc5Cer (GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide). It does not bind to many other glycosphingolipids without terminal N-acetylglucosamine residues that were tested. Thus, these data suggest that N-acetylglucosamine-terminated glycosphingolipids may serve as cell-surface attachment sites for mannose-binding protein in vivo. In addition, the binding specificity of the protein can be used as a sensitive probe for determining the levels of Lc3Cer and nLc5Cer in tissues, as it exhibits half-maximal binding to about 10 pmol of these lipids in solid phase assays, and detects less than 20 pmol of Lc3Cer in chromatogram overlay assays. This technique was utilized to demonstrate that one sample of chronic myeloid leukemia cells contains both Lc3Cer and nLc5Cer.

Gangliosides indirectly inhibit the binding of laminin to sulfatides

Laminin, a glycoprotein of basement membranes, agglutinates aldehyde-fixed erythrocytes. Laminin-mediated hemagglutination is strongly inhibited by some gangliosides and anionic phospholipids. Laminin, however, binds only to sulfatides among the lipids extracted from erythrocytes. We now report that gangliosides are remarkably potent inhibitors of laminin binding to sulfatides when both lipids are adsorbed on plastic. A 50% inhibition of laminin binding to 100 ng of sulfatides is obtained with 10 ng of GM3 and 8 ng of GM1, respectively. Mixing of sulfatides with neutral glycolipids, phosphatidyl choline, or cholesterol does not inhibit laminin binding, whereas mixing with sulfatide-depleted erythrocyte lipids enhances binding. Inhibition of binding by gangliosides is not due to competition for adsorption to the plastic, as preincubation of the adsorbed lipids with neuraminidase reverses inhibition by GM3, but not by GM1 which is not a substrate for the enzyme. These results are consistent with the observations that treatment of fixed erythrocytes with neuraminidase increases their agglutinability by laminin and that pretreatment of erythrocytes with gangliosides followed by washing gives similar inhibition as seen when gangliosides are present as competitive inhibitors. Thus, inhibition of laminin-mediated agglutination by gangliosides probably results from masking of erythrocyte sulfatides due to adsorption of gangliosides onto the membrane rather than from a direct competition for laminin binding sites.

A human Le(a−b+) blood group agglutinin prepared by polymerizing goat anti-lacto-N-difucohexaose I IgG

Abstract Anti-lacto-N-difucohexaose I IgG purified by affinity chromatography from the serum of a goat immunized with lacto-N-difucohexaose I coupled to polylysine does not agglutinate human erythrocytes. Upon polymerization of the antibody with dimethyl suberimidate, the higher oligomers at 0.1 μg/ml agglutinate erythrocytes from donors belonging to the Le (a − b +) blood group but not erythrocytes from donors belonging to the Le(a−b−) and Le(a+b−) blood groups. Oligomers smaller than pentamers are not agglutinins at up to 12 μg/ml.