Monoclonal antibodies that recognize the trisaccharide epitope Gal alpha 1-3Gal beta 1-4GlcNAc present on Ehrlich tumor cell membrane glycoproteins.
Monoclonal antibodies were prepared against the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc, 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 a Griffonia simplicifolia I (GS I) column which selectively binds alpha-D-galactosyl-terminated structures. Detection of Gal alpha 1-3Gal beta 1-4GlcNAc-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 Gal alpha 1-3Gal beta 1-4GlcNAc sequence. Inhibition of antibody-trisaccharide reactivity, examined employing an ELISA assay, revealed that two trisaccharides, Gal alpha 1-3Gal beta 1-4GlcNAc/Glc, were the best inhibitory haptens; Gal beta 1-4GlcNAc (LacNAc), Gal alpha 1-3Gal and Gal beta 1-4Glc (lactose) were poor inhibitors. Indirect immunofluorescence staining of unfixed Ehrlich cells using the monoclonal antibody at 4 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Takagaki
M
M
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0624.
Knibbs
R N
RN
Roth
J
J
Goldstein
I J
IJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D002240:Carbohydrate Sequence; D002286:Carcinoma, Ehrlich Tumor / Q000276:immunology* / Q000378:metabolism; D002417:Cattle; D002459:Cell Fusion; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005260:Female; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007150:Immunohistochemistry; D007797:Laminin / Q000302:isolation & purification; D008562:Membrane Glycoproteins / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D013954:Thyroglobulin / Q000276:immunology; D014312:Trisaccharides / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007797:Laminin; D008562:Membrane Glycoproteins; D014312:Trisaccharides; C076569:alpha-galactosyl epitope; D013954:Thyroglobulin