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

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Abstract
Two hybrid cell lines were prepared by fusion of mouse myeloma cells with spleen cells of BALB/c mice that had been immunized with the glycolipid ganglio-N-triosylceramide (asialo GM2). The specificity of monoclonal antibodies produced by these hybridomas, one an Ig[immunoglobulin]M and the other an IgG3, was 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-acetamido group of N-acetylgalactosamine, because oxidation with galactose oxidase produced a structure showing only minimal cross-reaction with the IgM. 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 glycolipid. 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 and with the native glycolipid. Neither antibody reacted significantly with any other natural glycolipids tested including several that were structurally related to asialo GM2 such as ganglioside GM2, ganglio-N-tetraosylceramide (asialo GM1) or ceramide dihexoside. In addition to the fine structure specificity described above, both antibodies apparently recognize the nonreducing terminal GalNAc.beta.1 .fwdarw. 4Gal structure. The strict antigenic specificity of these monoclonal anti-glycolipid antibodies indicates their great potential as specific probes for cell surface studies.