Preparation and characterization of antibody to galactosyl( α1 → 4)galactosyl( β1 → 4)glucosylceramide

• Published in: Biochimica et biophysica acta Vol. 582; no. 2; pp. 283 - 294
• Main Authors: Clarke, J.T.R., Embil, J.A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 18.01.1979
• Subjects: Animals; Antibodies - isolation & purification; Antibody Specificity; Cell Membrane - metabolism; Complement fixation; Cross Reactions; Globosides - immunology; Globosides - metabolism; Globotriaose antibody; Glycosphingolipid; Glycosphingolipids - immunology; Immunochemistry; Immunoglobulin M; Liposome; Rabbits; Gb 3, Gal( α1 → 4)Gal( β1 → 4)Glc → ceramide (globotriaosylceramide); Glycosphingolipid; globotetraosylceramide, GalNAc( β1 → 3)Gal( α1 → 4)Glc → ceramide; Liposome; Complement fixation; GM1 ganglioside, Gal β1 → 3GalNAc β1 → 4(NAcNeu α2 → 3)Gal β1 → 4Glc → ceramide; GM3 ganglioside, NAcNeu( α2 → 3) Gal( β1 → 4)Glc → ceramide; Globotriaose antibody
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(79)90391-X

Antiserum against galactosyl( α1 → 4)galactosyl( β1 → 4)glucosylceramide (globotriaosylceramide, Gb 3) was raised in rabbits by the administration of four weekly intramuscular injections of 1.5 mg of the purified glycolipid along with bovine serum albumin and Freund's complete adjuvant. AntiGb 3 activity was quantitated initially by immunoprecipitation employing Gb 3 mixed with 100-fold excess of lecithin and cholesterol (1 : 1 or 1 : 2, by wt.) as antigen. Subsequently, complement fixation tests done with antigen preparations containing Gb 3/lecithin/cholesterol (1 : 6 : 20, by wt.) showed antiGb 3 titres of up to 1 : 8192. Fractionation of the antiserum by BioGel A5m chromatography indicated the antibody was an IgM immunoglobulin. The partially purified antibody stimulated complement-dependent release of glucose from glucose-containing liposomes prepared with sphingomyelin/cholesterol/dicetylphosphate/Gb 3 (molar ratio, 100 : 75 : 11 : 1). The antibody crossreacted with the trisaccharide, Gal( α1 → 4)Gal( β1 → 4)Glc, but not with galactosylceramide, lactosylceramide, GM1 ganglioside, globotetraosylceramide, digalactosyldiglyceride or a number of low molecular weight saccharides.