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.
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References
Tamborrini M, Werz DB, Frey J et al (2006) Anti-carbohydrate antibodies for the detection of anthrax spores. Angew Chem Int Ed Engl 45:6581–6582
Tamborrini M, Oberli MA, Werz DB et al (2009) Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains. J Appl Microbiol 106:1618–1628
Tamborrini M, Holzer M, Seeberger PH et al (2010) Anthrax spore detection by a luminex assay based on monoclonal antibodies that recognize anthrose-containing oligosaccharides. Clin Vaccine Immunol 17:1446–1451
Anish C, Guo X, Wahlbrink A et al (2013) Plague detection by anti-carbohydrate antibodies. Angew Chem Int Ed Engl 52:9524–9528
Gao C, Liu Y, Zhang H et al (2014) Carbohydrate sequence of the prostate cancer-associated antigen F77 assigned by a mucin O-glycome designer array. J Biol Chem 289:16462–16477
Lee G, Cheung AP, Ge B et al (2012) CA215 and GnRH receptor as targets for cancer therapy. Cancer Immunol Immunother 61:1805–1817
Wang LX (2013) Synthetic carbohydrate antigens for HIV vaccine design. Curr Opin Chem Biol 17:997–1005
Lepenies B, Seeberger PH (2010) The promise of glycomics, glycan arrays and carbohydrate-based vaccines. Immunopharmacol Immunotoxicol 32:196–207
Oberli MA, Tamborrini M, Tsai YH et al (2010) Molecular analysis of carbohydrate-antibody interactions: case study using a Bacillus anthracis tetrasaccharide. J Am Chem Soc 132:10239–10241
Broecker F, Aretz J, Yang Y et al (2014) Epitope recognition of antibodies against a Yersinia pestis lipopolysaccharide trisaccharide component. ACS Chem Biol 9:867–873
Avery OT, Goebel WF (1931) Chemo-immunological studies on conjugated carbohydrate-proteins: V. The immunological specificity of an antigen prepared by combining the capsular polysaccharide of type III pneumococcus with foreign protein. J Exp Med 54:437–447
Jorge P, Abdul-Wajid A (1995) Sialyl-Tn-KLH, glycoconjugate analysis and stability by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Glycobiology 5:759–764
Borrow R, Dagan R, Zepp F et al (2011) Glycoconjugate vaccines and immune interactions, and implications for vaccination schedules. Expert Rev Vaccines 10:1621–1631
Oberli MA, Hecht ML, Bindschädler P et al (2011) A possible oligosaccharide-conjugate vaccine candidate for Clostridium difficile is antigenic and immunogenic. Chem Biol 18:580–588
Anish C, Martin CE, Wahlbrink A et al (2013) Immunogenicity and diagnostic potential of synthetic antigenic cell surface glycans of Leishmania. ACS Chem Biol 8:2412–2422
Martin CE, Broecker F, Oberli MA et al (2013) Immunological evaluation of a synthetic Clostridium difficile oligosaccharide conjugate vaccine candidate and identification of a minimal epitope. J Am Chem Soc 135:9713–9722
Eriksson M, Serna S, Maglinao M et al (2014) Biological evaluation of multivalent lewis X-MGL-1 interactions. Chembiochem 15:844–851
Wenzel T, Sparbier K, Mieruch T et al (2006) 2,5-Dihydroxyacetophenone: a matrix for highly sensitive matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of proteins using manual and automated preparation techniques. Rapid Commun Mass Spectrom 20:785–789
Yang Y, Oishi S, Martin CE et al (2013) Diversity-oriented synthesis of inner core oligosaccharides of the lipopolysaccharide of pathogenic Gram-negative bacteria. J Am Chem Soc 135:6262–6271
Mechetner E (2007) Development and characterization of mouse hybridomas. Methods Mol Biol 378:1–13
Reeves JP, Reeves PA (2001) Removal of lymphoid organs. Curr Protoc Immunol Chapter 1, Unit 1.9
Geissner A, Anish C, Seeberger PH (2014) Glycan arrays as tools for infectious disease research. Curr Opin Chem Biol 18:38–45
Moelling K, Broecker F, Kerrigan JE (2014) RNase H: specificity, mechanisms of action, and antiviral target. Methods Mol Biol 1087:71–84
Miron T, Wilchek M (1982) A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters. Anal Biochem 126:433–435
Diehl KH, Hull R, Morton D, European Federation of Pharmaceutical Industries Association and European Centre for the Validation of Alternative Methods et al (2001) A good practice guide to the administration of substances and removal of blood, including routes and volumes. J Appl Toxicol 21:15–23
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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Broecker, F., Anish, C., Seeberger, P.H. (2015). Generation of Monoclonal Antibodies against Defined Oligosaccharide Antigens. In: Lepenies, B. (eds) Carbohydrate-Based Vaccines. Methods in Molecular Biology, vol 1331. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2874-3_5
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DOI: https://doi.org/10.1007/978-1-4939-2874-3_5
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2873-6
Online ISBN: 978-1-4939-2874-3
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