title: High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization.
abstract: Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.
authors.affiliation: Department of. Neurology. Pharmacology. Neuroscience, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.
authors.forename: M P
authors.initials: MP
authors.lastname: Lunn
authors.forename: L A
authors.initials: LA
authors.lastname: Johnson
authors.forename: S E
authors.initials: SE
authors.lastname: Fromholt
authors.forename: S
authors.initials: S
authors.lastname: Itonori
authors.forename: J
authors.initials: J
authors.lastname: Huang
authors.forename: A A
authors.initials: AA
authors.lastname: Vyas
authors.forename: J E
authors.initials: JE
authors.lastname: Hildreth
authors.forename: J W
authors.initials: JW
authors.lastname: Griffin
authors.forename: R L
authors.initials: RL
authors.lastname: Schnaar
authors.forename: K A
authors.initials: KA
authors.lastname: Sheikh
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000915:Antibody Affinity; D002531:Cerebellum / Q000166:cytology; D005732:Gangliosides / Q000032:analysis* / Q000276:immunology*; D006433:Hemocyanins / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D007074:Immunoglobulin G / Q000276:immunology*; D007150:Immunohistochemistry; D051379:Mice; D018345:Mice, Knockout; D017350:N-Acetylgalactosaminyltransferases / Q000172:deficiency* / Q000235:genetics; D009474:Neurons / Q000737:chemistry; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D000911:Antibodies, Monoclonal; D005732:Gangliosides; D006433:Hemocyanins; D007074:Immunoglobulin G; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C021298:ganglioside, GD1a; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase; C032808:keyhole-limpet hemocyanin