Reactivity of two anti-galactosyl ceramide antibodies towards myelin basic protein.
Two anti-galactosyl ceramide antibodies (polyclonal Ab142 and a monoclonal antibody) were characterized in terms of their reactivity towards purified lipids and myelin basic protein. Polyclonal Ab142 is a rabbit anti-mouse galactosyl ceramide (Gal C) IgG. Antigenic recognition is dependent on both galactose and ceramide since neither could inhibit galactosyl ceramide binding by more than 10%. MAb-Gal C is a monoclonal antibody raised in mice against galactosyl ceramide. Binding of MAb-Gal C to Gal-C was equally inhibited by ceramide and galactose to approximately 50%, indicating that both groups are important for antibody recognition. MAb-Gal C was also shown to be reactive with the structurally related lipids, sphingomyelin and sulfatide. Polyclonal Ab142, although raised against Gal C, was shown to be 3-fold more reactive with component 8 (C-8) of myelin basic protein than Gal C. On the other hand, the MAb-Gal C which also reacted with C-8 was 2-fold less reactive with C-8 than with Gal C. Neither of these antibodies were reactive with component 1 (C-1) of myelin basic protein. An anti-MBP IgG was shown to be reactive with C-1 and C-8 but unreactive with Gal C. In competitive inhibition ELISA, C-8 was able to compete out 44% and 41% of Gal C binding to polyclonal Ab142 and MAb, respectively. The reverse competition demonstrated that Gal C could inhibit 75% of C-8 binding to both antibodies. D-galactose was unable to inhibit C-8 binding to either antibody, whereas ceramide was as efficient as Gal C.(ABSTRACT TRUNCATED AT 250 WORDS)
McLaurin
J
J
Department of Biochemistry, Hospital for Sick Children, Toronto, Ont., Canada.
Moscarello
M A
MA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D002518:Ceramides / Q000276:immunology; D002956:Citrulline / Q000276:immunology; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005690:Galactose / Q000276:immunology; D005699:Galactosylceramides / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D008081:Liposomes; D051379:Mice / Q000276:immunology; D004676:Myelin Basic Protein / Q000276:immunology*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011817:Rabbits / Q000276:immunology; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D002518:Ceramides; D000939:Epitopes; D005699:Galactosylceramides; D007074:Immunoglobulin G; D008081:Liposomes; D004676:Myelin Basic Protein; D010455:Peptides; D002956:Citrulline; D005690:Galactose