Abstract
One of the standard assays for the fungal pathogen Cryptococcus neoformans is the glucuronoxylomannan (GXM) ELISA. This assay utilizes monoclonal antibodies targeted against the critical virulence factor, the polysaccharide (PS) capsule. GXM ELISA is one of the most used assays in the field used for diagnosis of cryptococcal infection, quantification of PS content, and determination of binding specificity for antibodies. Here we present three variations of the GXM ELISA used by our group—indirect, capture, and competition ELISAs. We have also provided some history, perspective, and notes on these methods, which we hope will help the reader choose, and implement, the best assay for their research.
While it has long been referred to as the GXM ELISA, we also suggest a name update to better reflect our updated understanding of the polysaccharide antigens targeted by this assay. The Cryptococcal PS ELISA is a more accurate description of this set of methodologies and the antigens they measure. Finally, we discuss the limitations of this assay and put forth future plans for expanding the antigens assayed by ELISA.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
van de Moer A, Salhi SL, Cherniak R et al (1990) An anti-Cryptococcus neoformans monoclonal antibody directed against galactoxylomannan. Res Immunol 141:33–42
Bose I, Reese AJ, Ory JJ et al (2003) A yeast under cover: the capsule of Cryptococcus neoformans. Eukaryot Cell 2:655–663
Jesus MD, Nicola AM, Chow S-K et al (2010) Glucuronoxylomannan, galactoxylomannan, and mannoprotein occupy spatially separate and discrete regions in the capsule of Cryptococcus neoformans. Virulence 1:500–508
Dromer F, Salamero J, Contrepois A et al (1987) Production, characterization, and antibody specificity of a mouse monoclonal antibody reactive with Cryptococcus neoformans capsular polysaccharide. Infect Immun 55:742–748
Fromtling RA, Shadomy HJ (1982) Immunity in cryptococcosis: an overview. Mycopathologia 77:183–190
Dromer F, Charreire J, Contrepois A et al (1987) Protection of mice against experimental cryptococcosis by anti-Cryptococcus neoformans monoclonal antibody. Infect Immun 55:749–752
Casadevall A, Mukherjee J, Devi SJ et al (1992) Antibodies elicited by a Cryptococcus neoformans-tetanus toxoid conjugate vaccine have the same specificity as those elicited in infection. J Infect Dis 165:1086–1093
Cherniak R, Valafar H, Morris LC et al (1998) Cryptococcus neoformans chemoty** by quantitative analysis of 1H nuclear magnetic resonance spectra of glucuronoxylomannans with a computer-simulated artificial neural network. Clin Diagn Lab Immunol 5:146–159
Guazzelli L, Ulc R, Bowen A et al (2020) A synthetic glycan array containing Cryptococcus neoformans glucuronoxylomannan capsular polysaccharide fragments allows the map** of protective epitopes. Chem Sci 11:9209–9217
Crawford CJ, Guazzelli L, McConnell SA et al (2023) Synthetic glucuronoxylomannans enable molecular insights into antibody structure-function and fungal capsule architecture. ChemRxiv. Cambridge Open Engage, Cambridge
Todaro-Luck F, Reiss E, Cherniak R et al (1989) Characterization of Cryptococcus neoformans capsular glucuronoxylomannan polysaccharide with monoclonal antibodies. Infect Immun 57:3882–3887
Nussbaum G, Cleare W, Casadevall A et al (1997) Epitope location in the Cryptococcus neoformans capsule is a determinant of antibody efficacy. J Exp Med 185:685–694
Shapiro S, Beenhouwer DO, Feldmesser M et al (2002) Immunoglobulin G monoclonal antibodies to Cryptococcus neoformans protect mice deficient in complement component C3. Infect Immun 70:2598–2604
Ellerbroek P, Vecchiarelli A, Hoepelman A et al (2007) Immunology of infections with Cryptococcus neoformans. In: Brown GD, Netea MG (eds) Immunology of fungal infections. Springer Netherlands, Dordrecht, pp 383–407
Bowen A, Wear MP, Cordero RJB et al (2017) A monoclonal antibody to Cryptococcus neoformans glucuronoxylomannan manifests hydrolytic activity for both peptides and polysaccharides. J Biol Chem 292:417–434
Bowen A, Wear M, Casadevall A (2017) Antibody-mediated catalysis in infection and immunity. Infect Immun 85:e00202–e00217
Janda A, Eryilmaz E, Nakouzi A et al (2015) Variable region identical IgA and IgE to Cryptococcus neoformans capsular polysaccharide manifest specificity differences. J Biol Chem 290:12090–12100
Cordero RJB, Pontes B, Frases S et al (2013) Antibody binding to Cryptococcus neoformans impairs budding by altering capsular mechanical properties. J Immunol 190:317–323
McClelland EE, Nicola AM, Prados-Rosales R et al (2010) Ab binding alters gene expression in Cryptococcus neoformans and directly modulates fungal metabolism. J Clin Invest 120:1355–1361
Jiang A, Bryan RA, Morgenstern A et al (2012) Treatment of early and established Cryptococcus neoformans infection with radiolabeled antibodies in immunocompetent mice. Antimicrob Agents Chemother 56:552–554
Crawford CJ, Wear MP, Smith DFQ et al (2021) A glycan FRET assay for detection and characterization of catalytic antibodies to the Cryptococcus neoformans capsule. Proc Natl Acad Sci U S A 118:e2016198118
Mukherjee J, Casadevall A, Scharff MD (1993) Molecular characterization of the humoral responses to Cryptococcus neoformans infection and glucuronoxylomannan-tetanus toxoid conjugate immunization. J Exp Med 177:1105–1116
Wear MP, Hargett AA, Kelly JE et al (2022) Lyophilization induces alterations in cryptococcal exopolysaccharide resulting in reduced antibody binding. Carbohydr Polym 291:119547
Frases S, Nimrichter L, Viana NB et al (2008) Cryptococcus neoformans capsular polysaccharide and exopolysaccharide fractions manifest physical, chemical, and antigenic differences. Eukaryot Cell 7:319–327
Rodrigues ML, Nimrichter L, Oliveira DL et al (2007) Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport. Eukaryot Cell 6:48–59
Wear MP, Jacobs E, Wang S et al (2021) Cryptococcus neoformans capsule regrowth experiments reveal dynamics of enlargement and architecture. J Biol Chem 298:101769
Harlow E, Lane D (1988) Antibodies: a laboratory manual. CSHL Press, Cold Spring Harbor
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Wear, M.P., McConnell, S.A., Greengo, S.D., Lopes, L.L., Casadevall, A. (2024). Methods of Cryptococcal Polysaccharide Analysis Using ELISA. In: McClelland, E.E. (eds) Cryptococcus neoformans. Methods in Molecular Biology, vol 2775. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3722-7_16
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3722-7_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3721-0
Online ISBN: 978-1-0716-3722-7
eBook Packages: Springer Protocols