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. 1979 Oct 1;150(4):1008–1019. doi: 10.1084/jem.150.4.1008

Production of monoclonal antibodies specific for two distinct steric portions of the glycolipid ganglio-N-triosylceramide (asialo GM2)

PMCID: PMC2185672  PMID: 512581

Abstract

Two hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of BALB/c mice that had been immunized with the glycolipid ganglio-N-triosylceramide (asialo GM2). The specificity of the monoclonal antibodies produced by these hybridomas, one an IgM and the other an IgG3, has been defined by hemagglutination inhibition, complement fixation, and lysis of glycolipid liposomes by antibody and complement. A major determinant recognized by the IgM antibody is the nonreducing terminal N-acetylgalactosamine including the C6 primary hydroxyl group, but excluding the C2-acetamide group of N- acetylgalactosamine, because oxidation with galactose oxidase produced a structure showing only minimal cross-reaction with the IgM but replacement of the N-acetyl group with an N-n-butyryl group produced a glycolipid that reacts with IgM antibody to the same extent as with the unmodified glycoplipd. A major determinant recognized by the IgG3 antibody is the terminal N-acetylgalactosamine including the C2- acetamido group, but excluding the C6 primary hydroxyl group of N- acetylgalactosamine, because replacement of the N-acetyl group with an N-n-butyryl group produced a glycolipid that did not react with the IgG3 antibody; in striking contrast the IgG3 antibody reacted with the C6-oxidized glycolipid as well as with the native glycolipid. Neither antibody reacted significantly with any other natural glycolipids tested including several that are structurally related to asialo GM2 such as ganglioside GM2, ganglio-N-tetraosylceramide (asialo GM1), or ceramide dihexoside. These results indicated that in addition to the fine structure specificity described above both antibodies recognize the nonreducing terminal GalNAc beta 1 leads to 4Gal structure. The strict antigenic specificity of these monoclonal anti-glycolipid antibodies indicates their great potential as specific probes for cell surface studies.

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Selected References

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  1. Briles D. E., Davie J. M. Detection of isoelectric focused antibody by autoradiography and hemolysis of antigen-coated erythrocytes. A comparison of methods. J Immunol Methods. 1975 Oct;8(4):363–372. doi: 10.1016/0022-1759(75)90058-7. [DOI] [PubMed] [Google Scholar]
  2. Feizi T., Childs R. A., Watanabe K., Hakomori S. I. Three types of blood group I specificity among monoclonal anti-I autoantibodies revealed by analogues of a branched erythrocyte glycolipid. J Exp Med. 1979 Apr 1;149(4):975–980. doi: 10.1084/jem.149.4.975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fishman P. H., Brady R. O. Biosynthesis and function of gangliosides. Science. 1976 Nov 26;194(4268):906–915. doi: 10.1126/science.185697. [DOI] [PubMed] [Google Scholar]
  4. Galanos C., Lüderitz O., Westphal O. Preparation and properties of antisera against the lipid-A component of bacterial lipopolysaccharides. Eur J Biochem. 1971 Dec 22;24(1):116–122. doi: 10.1111/j.1432-1033.1971.tb19661.x. [DOI] [PubMed] [Google Scholar]
  5. Grey H. M., Hirst J. W., Cohn M. A new mouse immunoglobulin: IgG3. J Exp Med. 1971 Feb 1;133(2):289–304. doi: 10.1084/jem.133.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hakomori S. I., Teather C., Andrews H. Organizational difference of cell surface "hematoside" in normal and virally transformed cells. Biochem Biophys Res Commun. 1968 Nov 25;33(4):563–568. doi: 10.1016/0006-291x(68)90332-x. [DOI] [PubMed] [Google Scholar]
  7. Hakomori S. Structures and organization of cell surface glycolipids dependency on cell growth and malignant transformation. Biochim Biophys Acta. 1975 Mar 20;417(1):55–89. doi: 10.1016/0304-419x(75)90008-6. [DOI] [PubMed] [Google Scholar]
  8. Holmgren J. Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid. Infect Immun. 1973 Dec;8(6):851–859. doi: 10.1128/iai.8.6.851-859.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  10. Köhler G., Milstein C. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur J Immunol. 1976 Jul;6(7):511–519. doi: 10.1002/eji.1830060713. [DOI] [PubMed] [Google Scholar]
  11. Laine R. A., Yoggeswaran G., Hakorori S. Glycosphingolipids covalently linked to agarose gel or glass beads. Use of the compounds for purification of antibodies directed against globoside and hematoside. J Biol Chem. 1974 Jul 25;249(14):4460–4466. [PubMed] [Google Scholar]
  12. Lingwood C. A., Hakomori S. Selective inhibition of cell growth and associated changes in glycolipid metabolism induced by monovalent antibodies to glycolipids. Exp Cell Res. 1977 Sep;108(2):385–391. doi: 10.1016/s0014-4827(77)80045-1. [DOI] [PubMed] [Google Scholar]
  13. Lingwood C. A., Ng A., Hakomori S. Monovalent antibodies directed to transformation-sensitive membrane components inhibit the process of viral transformation. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6049–6053. doi: 10.1073/pnas.75.12.6049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Marcus D. M., Janis R. Localization of glycosphingolipids in human tissues by immunofluorescence. J Immunol. 1970 Jun;104(6):1530–1539. [PubMed] [Google Scholar]
  15. Marcus D. M., Schwarting G. A. Immunochemical properties of glycolipids and phospholipids. Adv Immunol. 1976;23:203–240. [PubMed] [Google Scholar]
  16. Niemann H., Watanabe K., Hakomori S. Blood group i and I activities of "lacto-N-norhexaosylceramide" and its analogues: the structural requirements for i-specificities. Biochem Biophys Res Commun. 1978 Apr 28;81(4):1286–1293. doi: 10.1016/0006-291x(78)91275-5. [DOI] [PubMed] [Google Scholar]
  17. Nowinski R. C., Lostrom M. E., Tam M. R., Stone M. R., Burnette W. N. The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of ecotropic murine leukemia viruses. Virology. 1979 Feb;93(1):111–126. doi: 10.1016/0042-6822(79)90280-0. [DOI] [PubMed] [Google Scholar]
  18. Perlmutter R. M., Hansburg D., Briles D. E., Nicolotti R. A., Davie J. M. Subclass restriction of murine anti-carbohydrate antibodies. J Immunol. 1978 Aug;121(2):566–572. [PubMed] [Google Scholar]
  19. Rosenfelder G., Young W. W., Jr, Hakomori S. I. Association of the glycolipid pattern with antigenic alterations in mouse fibroblasts transformed by murine sarcoma virus. Cancer Res. 1977 May;37(5):1333–1339. [PubMed] [Google Scholar]
  20. Seyama Y., Yamakawa T. Chemical structure of glycolipid of guinea pig red blood cell membrane. J Biochem. 1974 Apr;75(4):837–842. doi: 10.1093/oxfordjournals.jbchem.a130455. [DOI] [PubMed] [Google Scholar]
  21. Six H. R., Young W. W., Jr, Uemura K., Kinsky S. C. Effect of antibody-complement on multiple vs. single compartment liposomes. Application of a fluorometric assay for following changes in liposomal permeability. Biochemistry. 1974 Sep 10;13(19):4050–4058. doi: 10.1021/bi00716a037. [DOI] [PubMed] [Google Scholar]
  22. Suzuki Y., Suzuki K. Specific radioactive labeling of terminal n-acetylgalactosamine of glycosphingolipids by the galactose oxidase-sodium borohydride method. J Lipid Res. 1972 Sep;13(5):687–690. [PubMed] [Google Scholar]
  23. Swanstrom R., Shank P. R. X-Ray Intensifying Screens Greatly Enhance the Detection by Autoradiography of the Radioactive Isotopes 32P and 125I. Anal Biochem. 1978 May;86(1):184–192. doi: 10.1016/0003-2697(78)90333-0. [DOI] [PubMed] [Google Scholar]
  24. Udenfriend S., Stein S., Böhlen P., Dairman W., Leimgruber W., Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972 Nov 24;178(4063):871–872. doi: 10.1126/science.178.4063.871. [DOI] [PubMed] [Google Scholar]
  25. Watanabe K., Hakomori S. I., Childs R. A., Feizi T. Characterization of a blood group I-active ganglioside. Structural requirements for I and i specificities. J Biol Chem. 1979 May 10;254(9):3221–3228. [PubMed] [Google Scholar]
  26. Wolf B. A., Robbins P. W. Cell mitotic cycle synthesis of NIL hamster glycolipids including the Forssman antigen. J Cell Biol. 1974 Jun;61(3):676–687. doi: 10.1083/jcb.61.3.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zopf D. A., Ginsburg A., Ginsburg V. Goat antibody directed against a human Leb blood group hapten, lacto-N-difucohexaose I. J Immunol. 1975 Dec;115(6):1525–1529. [PubMed] [Google Scholar]

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