Immunoglobulin G specifically binding plant N-glycans with high affinity could be generated in rabbits but not in mice

• Published in: Glycobiology (Oxford) Vol. 16; no. 4; pp. 349 - 357
• Main Authors: Jin, Chunsheng, Bencúrová, Monika, Borth, Nicole, Ferko, Boris, Jensen-Jarolim, Erika, Altmann, Friedrich, Hantusch, Brigitte
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-04-2006; Oxford Publishing Limited (England)
• Subjects: Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Antibody Affinity - immunology; Antibody Specificity - immunology; binding affinity; bovine serum albumin; BSA; carbohydrate epitope; CCD; cross-reactive carbohydrate determinant; glycoprotein; Glycoproteins - chemistry; Glycoproteins - immunology; HAT; horseradish peroxidase; HRP; hypoxanthine-aminopterin- thymidine; immunogenic N-glycan; Immunoglobulin G - chemistry; Immunoglobulin G - immunology; Mice; Oligosaccharides, Branched-Chain - chemistry; Oligosaccharides, Branched-Chain - immunology; Plant Proteins - chemistry; Plant Proteins - immunology; Plants - chemistry; Plants - immunology; Rabbits; Species Specificity; SPR; Surface Plasmon Resonance
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwj071

Xylosylated and core [alpha]1,3-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 [alpha]1,3-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.