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Generation of Monoclonal Antibodies against Defined Oligosaccharide Antigens

  • Felix Broecker
  • Chakkumkal AnishEmail author
  • Peter H. SeebergerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1331)

Abstract

Unique carbohydrate antigens are expressed on the surface of various pathogens, including bacteria, parasites, and viruses, and aberrant glycosylation is a frequent feature of cancer cells. Antibodies recognizing such carbohydrate antigens may be used for the specific detection of potentially harmful cells, immunohistochemistry, and diagnostic and therapeutic applications. The generation of specific and strongly binding antibodies against defined carbohydrate epitopes is challenging, since isolated carbohydrates often suffer from low purity, usually have limited immunogenicity, and induce antibodies of low affinity. We describe a protocol to generate highly affine monoclonal antibodies (mAbs) against pure and defined synthetic carbohydrate antigens. First, an oligosaccharide is covalently coupled to an immunogenic carrier protein to obtain a glycoconjugate. This glycoconjugate is used to raise oligosaccharide-specific antibodies in mice, and splenocytes are fused with myeloma cells to form hybridomas. Hybridoma clones producing oligosaccharide-specific mAbs are selected by glycan microarray screening. Selected clones are expanded and mAbs are purified from the cell culture supernatant. This protocol is suitable to procure carbohydrate-specific mAbs of high specificity, selectivity, and affinity that may be useful for a variety of biochemical and medical applications.

Key words

Antigen Carbohydrate Epitope Glycan Glycoconjugate Hybridoma mAb Monoclonal antibody Oligosaccharide Pathogen detection 

Notes

Acknowledgements

We thank Dr. You Yang for providing the Kdo antigen and Pfénex, Inc. for providing CRM197 at a reduced price for academic institutions. We acknowledge careful and critical reviewing of the manuscript by Andreas Geissner, Anika Reinhardt, Benjamin Schumann, and Stefan Matthies. We thank the Max Planck Society, the Körber Foundation (Körber Prize to PHS), and the German Federal Ministry of Education and Research (grant No. 0315447) for generous financial support.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of BiomolecularSystemsMax Planck Institute of Colloids and InterfacesPotsdamGermany
  2. 2.Freie Universität Berlin, Institute of Chemistry and BiochemistryBerlinGermany
  3. 3.Bacterial Vaccines Discovery and Early DevelopmentJanssen Pharmaceuticals (Johnson & Johnson)LeidenThe Netherlands
  4. 4.Department of Biomolecular SystemsMax Planck Institute of Colloids and InterfacesPotsdamGermany

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