Convenient preparation and characterization of a monoclonal antibody for the N-linked sugar chain of a glycoprotein using a microbial endoglycosidase.
We attempted to obtain the monoclonal antibody specific for the N-linked complex-type sialo-oligosaccharide in glycoproteins. We first synthesized a chimeric immunoantigen having an N-linked complex-type of oligosaccharide of glycopeptide, which was bound to a p-formylphenyl compound and conjugated with phosphatidylethanolamine dimyristoyl using the transglycosylation activity of a microbial endoglycosidase (Endo-M) and a reductive amination reaction. This preparative method was convenient and provided a good yield. By immunizing mice with this chimeric neoglycolipid, the monoclonal antibody for the complex-type of sialo-oligosaccharide was obtained in the culture fluid of the cell line even though it was relatively unstable. The monoclonal antibody reacted with various glycoproteins having complex-type sialo-oligosaccharides, but not with those having complex-type asialo-oligosaccharides and high mannose types of oligosaccharides, or with any glycosphingolipids. One of epitopes of this monoclonal antibody seemed to be an alpha-2,6-linked sialic acid at the non-reducing end of the sialo-oligosaccharide of the glycoprotein.
Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
Daisuke
D
Murakami
Yoshitaka
Y
Shimada
Satomi
S
Kamiya
Kohji
K
Yamazaki
Yutaka
Y
Makimura
Kazuo
K
Ito
Noshi
N
Minamiura
Kenji
K
Yamamoto
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D002478:Cells, Cultured; D005260:Female; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D009089:Mucor / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D006026:Glycoside Hydrolases