Immunoglobulin G specifically binding plant N-glycans with high affinity could be generated in rabbits but not in mice.
Xylosylated and core alpha1,3-fucosylated N-glycans from plants are immunogenic, and they play a still obscure role in allergy and in the field of plant-made protein pharmaceuticals. We immunized mice to generate monoclonal antibodies (mAbs) binding plant N-glycans specifically via the epitope containing either the xylose or the core alpha1,3-fucose residue. Splenocytes expressing N-glycan-specific antibodies derived from C57BL/6 mice previously immunized with plant glycoproteins were preselected by cell sorting to generate hybridoma lines producing specific antibodies. However, we obtained only mAbs unable to distinguish fucosylated from xylosylated N-glycans and reactive even with the pentasaccharide core Man3GlcNAc2. In contrast, immunization of rabbits yielded polyclonal sera selectively reactive with either fucosylated or xylosylated N-glycans. Purification of these sera using glyco-modified neoglycoproteins coupled to a chromatography matrix provided polyclonal sera suitable for affinity determination. Surface plasmon resonance measurements using sensor chips with immobilized glyco-modified transferrins revealed dissociation constants of around 10(-9) M. This unexpectedly high affinity of IgG antibodies toward carbohydrate epitopes has repercussions on our conception of the binding strength and significance of antiglycan IgE antibodies in allergy.
Jin
Chunsheng
C
Department of Chemistry, University of Natural Resources and Applies Life Sciences (BOKU), 1190 Vienna, Austria.
Bencúrová
Monika
M
Borth
Nicole
N
Ferko
Boris
B
Jensen-Jarolim
Erika
E
Altmann
Friedrich
F
Hantusch
Brigitte
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D025942:Oligosaccharides, Branched-Chain / Q000737:chemistry* / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology; D010944:Plants / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits; D013045:Species Specificity; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D007074:Immunoglobulin G; D025942:Oligosaccharides, Branched-Chain; D010940:Plant Proteins