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Abstract
Monoclonal antibody 101 produced by a hybridoma obtained by fusion with NS-1 myeloma cells of spleen cells from a mouse immunized with the human epidermoid carcinoma cell line, A431, specifically precipitates epidermal growth factor receptor, a glycoprotein of 170,000 Mr solubilized from A431 cell membranes (Richert, N. D., Willingham, M. C., and Pastan, I. H. (1983) J. Biol. Chem. 258, 8902-8907). The antibody also binds to neutral glycolipids extracted from A431 cells as evidenced by solid phase radioimmunoassay and by autoradiography. Binding of antibody to its target is inhibited by lacto-N-fucopentaose I but not by 2'-fucosyllactose or related oligosaccharides. Thus, antibody 101 is probably directed against the human blood group H type 1 sugar sequence Fuc alpha 1-2Gal beta 1-3GlcNAc . . .. This sequence presumably occurs on the epidermal growth factor receptor. Monoclonal antibody 102 produced by another hybridoma from the same fusion has the same cell specificity as antibody 101 and also binds to neutral glycolipids. However, binding of antibody 102 to its target is inhibited by 2'-fucosyllactose and not by lacto-N-fucopentaose I or related oligosaccharides. Thus, antibody 102 is probably directed against the human blood group H type 2 sugar sequence Fuc alpha 1-2Gal beta 1-4GlcNAc . . .. Antibody 102 does not precipitate solubilized epidermal growth factor receptor. Both antibodies bind to neutral glycolipids extracted from human erythrocytes belonging to blood group O but not to neutral glycolipids extracted from human erythrocytes with the "Bombay" phenotype.
Highlights
Of 170,000 M, solubilized from A431 cell membranes Antibody 101 does not block the binding of EGF nor does it
Maximum binding of antibody 102 is achieved with glycolipids from approximately 0.6 pg of cells whereas binding of antibody 101 is still increasing with glycolipids from approximately 60 pg of cells (Fig. 1).Neither antibody binds to gangliosides extracted from A431 cells or KB cells in solid phase radioimmunoassay
Neither antibody detects antigens in glycolipids from Bombay erythrocytes,suggesting that bothare directed against the human blood group H antigen which is missing in the Bombay phenotype [5].The large diffuse spots asopposed to bands seen in lanes 4, 9, IO, and 14 represent nonspecific binding of antibody to lipid material and oftenoccur when large quantities of extracts are chromatographed
Summary
Materials-2’-Fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, To obtain monoclonal antibodiesagainsttheepidermal growth factorreceptor,Balb/c mice were immunizedwith A431 cells,a human epidermoid carcinoma cell line. After fusion of the mouse spleen cells with NS-1 myeloma cells, a hybridoma was obtained thatproduces an IgG antibody (antibody 101) whichspecifically precipitates thesolubilized EGF recepattle,WA).Otherneutral glycolipids were isolatedfromvarious tissues [8].For structures see Table I. Affinity-purified goat antibodies to mouse IgM (G)and mouse IgG (y) were purchased from Kirkegaard and Perry .Laboratories, Inc. Evidence that antibody 101 recognizes and rhodamine-labeled goat anti-mouse antisera (Cappel Laboratories, Westchester, PA) were affinity-purified on a column of mouse. Cells-The A431 human epidermoid carcinoma cell line was obtained from Dr George Todaro (NIH). KB cells were obtained from the American TypeCulture Collection(Rockville, MD).
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