Immunochemical studies of conjugates of isomaltosyl oligosaccharides to lipid: production and characterization of mouse hybridoma antibodies specific for stearyl-isomaltosyl oligosaccharides.
Twelve C57BL/6J hybridoma clones, 9, 2 and 1 from mice immunized with stearyl-isomaltotetraose, stearyl-isomaltopentaose and stearyl-isomaltohexaose respectively were characterized. Seven produced IgA and 5 IgM. The specificities and sizes of their combining sites were determined by quantitative precipitin and precipitin inhibition assays. All 12 hybridoma antibodies precipitated with alpha 1----6 dextran B512 and linear dextran LD7, indicating that they recognize an internal -Glc alpha 1----6Glc alpha 1----6Glc- determinant. This in contrast with the results with rabbit antisera obtained in response to the same immunogen which recognize the non-reducing terminal determinant Glc alpha 1----6Glc alpha 1----6Glc-. Of the 12 hybridoma antibodies, 1 has an antibody combining site complementary to 4 alpha 1----6-linked glucoses while others have combining sites complementary to isomaltohexaose or isomaltoheptaose. The large combining-site sizes found in C57BL/6 hybridoma clones may be related to the pre-existing clonal repertoire in this strain. Binding constants of monomers of these antibodies for dextran B512 and isomaltoheptaose determined by affinity electrophoresis range from 1.4 X 10(3) to 4.6 X 10(5) ml/g and from 1.2 X 10(3) to 3.5 X 10(4) M-1 respectively, which is consistent with previous studies in the anti-dextran B512 system. The use of synthetic glycolipids as antigens enables us to study the gene control of antibody responses to glycolipids and to investigate the combining-site specificities of antibodies to a single antigenic determinant. Results so far show that all 12 hybridoma proteins are different despite the simplicity of the antigens. The findings provide further insight into the specificity of antibody combining sites.
E
E
Lai
E A
EA
Kabat
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001666:Binding Sites, Antibody; D003911:Dextrans / Q000276:immunology; D006017:Glycolipids / Q000276:immunology; D007070:Immunoglobulin A / Q000096:biosynthesis; D007075:Immunoglobulin M / Q000096:biosynthesis; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D009844:Oligosaccharides / Q000276:immunology*; D011233:Precipitin Tests; D013229:Stearic Acids / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000941:Antigens; D003911:Dextrans; D006017:Glycolipids; D007070:Immunoglobulin A; D007075:Immunoglobulin M; D009844:Oligosaccharides; D013229:Stearic Acids; C031488:stearyl-isomaltosyl oligosaccharide; C047512:isomaltotetraose; C047511:isomaltopentaose; C024046:isomaltohexose