Abstract
Monoclonal antibodies were prepared against the trisaccharide Galα1-3Galβ1-4GlcNAc, a sequence which occurs on the surface of Ehrlich ascites tumor cells as well as in thyroglobulin, laminin and a variety of other proteins. This was accomplished by immunizing BALB/c mice with the fraction of Ehrlich cell membrane glycoproteins obtained by affinity chromatography on aGriffonia simplicifolia I (GS I) column which selectively binds α-d-galactosyl-terminated structures. Detection of Galα1-3Galβ1-4GlcNAc-specific antibodies was accomplished by employing glycoproteins containing the trisaccharide sequence; fusion with spleen cells from an immunized mouse was accomplished in the presence of polyethylene glycol (PEG1500). An enzyme-linked immunosorbent assay (ELISA) system was used to identify two clones (2.10G and 6.8E), which recognized the desired trisaccharide conjugate. These clones also recognized a thyroglobulin fraction isolated by GS I affinity chromatography and murine laminin, both of which possess the Galα1-3Galβ1-4GlcNAc sequence. Inhibition of antibody-trisaccharide reactivity, examined employing an ELISA assay, revealed that two trisaccharides, Galα1-3Galβ1-4GlcNAc/Glc, were the best inhibitory haptens; Galβ1-4GlcNAc (LacNAc), Galα1-3Gal and Galβ1-4Glc (lactose) were poor inhibitors. Indirect immunofluorescence staining of unfixed Ehrlich cells using the monoclonal antibody at 4° C revealed fluorescence over the entire cell surface. Indirect immunogold labeling of semithin and ultrathin sections of aldehyde fixed and Lowicryl K4M-embedded Ehrlich cells resulted in specific labeling of the cell surface and internal structure. Immunoblot analysis revealed that removal of the α-galactosyl residues of laminin by α-galactosidase abolished reactivity with the monoclonal antibodies. The availability of this antibody, which belongs to the IgM family of immunoglobulins, now makes possible the detection of this sugar sequence on cells and tissue sections, as well as on glycoproteins in solution.
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References
Avila JL, Rojas M, Galili U (1989) Immunogenic Galα1–3Gal carbohydrate epitopes are present on pathogenic americanTrypanosoma andLeishmania. J Immunol 142:2828–2834
Castronovo V, Foidart JM, Li Vecchi M, Foidart JB, Bracke M, Mareel M, Mahieu P (1987) Human anti-α-galactosyl IgG reduces the lung colonization by murine MO4 cells. Invasion Metastasis 7:325–345
Castronovo V, Colin C, Parent B, Foidart M Jr, Lambotte R, Mahieu P (1989) Possible role of human natural anti-Gal antibodies in the natural antitumor defence system. J Natl Cancer Inst 81:212–216
Cummings RD, Kornfeld S (1982) Characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilizedPhaseolus vulgaris leukoagglitinating and erythroagglutinating lectins. J Biol Chem 257:11230–11234
Cummings RD, Kornfeld S (1984) The distribution of repeating [Galβ1, 4GlcNacβ1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BM5147 and PHAR2.1. J Biol Chem 259:6253–6260
Davin JC, Malaise M, Foidart J, Mahieu P (1987) Anti-α-galactosyl antibodies and immune complexes in children with Henoch-Schonlein purpura or IgA nephropathy. Kidney International 31:1132–1139
Dorland L, Van Halbeek H, Vliegenthart JFG (1984) The identification of terminal α(1–3)-linked galactose inN-acetyl-lactosamine type of glycopeptides by means of 500-MHz 1H-NMR spectroscopy. Biochem Biophys Res Commun 122:859–866
Eckhardt AE, Goldstein IJ (1979) Isolation and characterization of a family of α-d-galactosyl-containing glycopeptides from Ehrlich ascites tumor cells. In Gregory JD, Jeanloz RW (eds) Glycoconjugate research, vol 2. Academic Press, New York, pp 1043–1045
Eckhardt AE, Goldstein IJ (1983a) Occurrence of α-d-galactosyl glycoproteins on Ehrlich tumor cell membranes. Biochemistry 22:5280–5289
Eckhardt AE, Goldstein IJ (1983b) Isolation and characterization of a family of α-d-galactosyl-containing glycopeptides from Ehrlich ascites tumor cells. Biochemistry 22:5290–5297
Edge ASB, Spiro RG (1985) Thyroid cell surface glycoproteins. J Biol Chem 260:15332–15338
Galfre G, Howe SC, Milstein C, Butcher GW, Howard JC (1977) Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266:530–552
Galili U, Rachmilewitz EA, Peleg A, Flechner I (1984) A unique natural human IgG antibody with anti-α-galactosyl reactivity. J Exp Med 160:1519–1531
Galili U, Macher BA, Buehler J, Shohet SB (1985a) Human natural anti-α-galactosyl IgG. II. Specific recognition of α(1–3)-linked galactose residues. J Exp Med 162:573–582
Galili U, Buehler J, Shohet SB, Macher BA (1985b) The human natural anti-Gal IgG. III. The subtlety of immune tolerance in man as demonstrated by crossreactivity between natural anti-Gal and anti-B antibodies. J Exp Med 165:693–704
Galili U, Basbaum CB, Shohet SB, Buehler J, Macher BA (1987) Identification of erythrocyte Galα1–3Gal glycosphingolipids with a mouse monoclonal antibody, Gal-13. J Biol Chem 262:4683–4688
Galili U, Shohet SB, Kobrin E, Stults CLM, Macher BA (1988) Man, apes and Old World monkeys differ from other mammals in the expression of α-galactosyl epitopes on nucleated cells. J Biol Chem 263:17755–17762
Gil J, Alvarez R, Vinuela JE, Ruiz de Morales JG, Bustos A, De la Concha EG, Subiza JL (1990) Inhibition of in vivo tumor growth by a monoclonal IgM antibody recognizing tumor cell surface carbohydrates. Cancer Res 50:7301–7306
Hudson HL, Hay FC (1976) Affinity chromatography. In: Practical immunology. Blackwell, Oxford, pp 203–225
Knibbs RN, Perini F, Goldstein IJ (1989) Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin. Biochemistry 28:6379–6392
Lapresle C, Goldstein IJ (1969) Immunogenicity of a fragment of human serum albumin. J Immunol 102:733–742
Lapresle C, Webb T (1965) Isolation and study of a fragment of human serum albumin containing one of the antigenic sites of the whole molecule. Biochem J 95:245–251
Lucocq JM, Baschong W (1986) Preparation of protein colloidal gold complexes in the presence of commonly used buffers. Eur J Cell Biol 42:332–337
McCoy JP, Lloyd RV, Wicha MS, Varani J (1984) Identification of a laminin-like substance on the surface of high-malignant murine fibrosarcoma cells. J Cell Sci 65:139–151
McKearn TJ (1980) Fusion of cells in an adherent monolayer. In Kennet RH, McKearn TJ, Bechtol KB (eds) Monoclonal antibodies, hybridomas: a new dimension in biological analysis. Plenum Press, New York, pp 368–369
Milani SR, Travassos LR (1988) Anti-α-galactosyl antibodies in chagasic patients. Possible biological significance. Braz J Med Biol Res 21:1275–1286
Orkin RW, Gehron P, McGoodwin EB, Martin GR, Valentine T, Swarm R (1977) A murine tumor producing a matrix of basement membrane. J Exp Med 145:204–220
Peters BP, Goldstein IJ (1979) The use of fluorescein-conjugatedBandeiraea simplicifolia B4-isolectin as a histochemical reagent for the detection of α-d-galactopyranosyl groups. Exp Cell Res 120:321–334
Randle BJ (1982) Cosegregation of monoclonal antibody reactivity and cell behaviour in the mouse preimplantation embryo. J Embryol Exp Morphol 70:261–278
Randle BJ (1983) Lineage and non-lineage related expression of an anti-teratocarcinoma monoclonal antibody reactivity in the post-implantation embryo and adult mouse. J Reprod Immunol 5:101–114
Roth J (1989) Postembedding labeling on Lowicryl K4M tissue sections: detection and modification of cellular components. Methods Cell Biol 31:513–551
Roth J, Bendayan M, Carlemalm E, Villiger W, Garavito M (1981) Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J Histochem Cytochem 29:663–671
Santer UV, De Santis R, Hard KJ, Kuik JA van, Vliegenthart J FG, Won B Glick MC (1989) N-linked oligosaccharide changes with oncogenic transformation require sialylation of multiantennae. Eur J Biochem 181:249–260
Shibata S, Peters B, Roberts D, Goldstein IJ, Liotta LA (1982) Isolation of laminin by affinity chromatography on immobilizedGriffonia simplicifolia I lectin. FEBS Lett 142:194–198
Shibuya N, Berry JE, Goldstein IJ (1988) One-step purification of murine IgM and human α2-macroglobulin by affinity chromatography on immobilized showdrop bulb lectin. Arch Biochem Biophys 267:676–680
Spiro RG, Bhoyroo VD (1984) Occurrence of α-d-galactosyl residues in the thyroglobulins from several species. J Biol Chem 259:9858–9866
Thall A, Galili U (1990) Distribution of Galα1–3Galβ1–4GlcNAc residues on secreted mammalian glycoproteins (thyroglobulin, fibrinogen and immunoglobulin G) as measured by a sensitive solid-phase radioimmunoassay. Biochemistry 29:3959–3965
Varani J, Lovett EJ, McCoy JP, Shibata S, Maddox DE, Goldstein IJ, Wicha M (1983a) Differential expression of a laminin-like substance by high- and low-metastatic tumor cells. Am J Pathol 11:27–34
Varani J, Lovett EJ, Wicha M, Malinoff H, McCoy JP (1983b) Cell surface α-d-galactosyl end group: use as a marker in the isolation of tumor cell lines with varying cancer-causing potential. J Natl Cancer Inst 71:1281–1287
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Takagaki, M., Knibbs, R.N., Roth, J. et al. Monoclonal antibodies that recognize the trisaccharide epitope Galα1-3Galβ1-4GlcNAc present on Ehrlich tumor cell membrane glycoproteins. Histochemistry 100, 139–147 (1993). https://doi.org/10.1007/BF00572900
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DOI: https://doi.org/10.1007/BF00572900