Monoclonal Antibody AP3 Binds Galactomannan Antigens Displayed by the Pathogens Aspergillus flavus, A. fumigatus, and A. parasiticus.

Max Schubert,Vadim B. Krylov,Sheng Xue,Nikolay E. Nifantiev,Stefan Schillberg,Frank Ebel,Greta Nölke,Annegret Vaggelas,Ivana Chudobová

doi:10.3389/fcimb.2019.00234

Abstract

Aspergillus fumigatus and A. flavus are the fungal pathogens responsible for most cases of invasive aspergillosis (IA). Early detection of the circulating antigen galactomannan (GM) in serum allows the prompt application of effective antifungal therapy, thus improving the survival rate of IA patients. However, the use of monoclonal antibodies (mAbs) for the diagnosis of IA is often associated with false positives due to cross-reaction with bacterial polysaccharides. More specific antibodies are therefore needed. Here we describe the characterization of the Aspergillus-specific mAb AP3 (IgG1κ), including the precise identification of its corresponding antigen. The antibody was generated using A. parasiticus cell wall fragments and was shown to bind several Aspergillus species. Immunofluorescence microscopy revealed that AP3 binds a cell wall antigen, but immunoprecipitation and enzyme-linked immunosorbent assays showed that the antigen is also secreted into the culture medium. The inability of AP3 to bind the A. fumigatus galactofuranose (Galf )-deficient mutant ΔglfA confirmed that Galf residues are part of the epitope. Several lines of evidence strongly indicated that AP3 recognizes the Galf residues of O-linked glycans on Aspergillus proteins. Glycoarray analysis revealed that AP3 recognizes oligo-[β-D-Galf-1,5] sequences containing four or more residues with longer chains more efficiently. We also showed that AP3 captures GM in serum, suggesting it may be useful as a diagnostic tool for patients with IA.

Highlights

The genus Aspergillus comprises 339 filamentous fungi that are ubiquitous in nature and have many potential applications in biotechnology, but some species pose a risk to human and animal health (Samson et al, 2014)
Pure cultures of A. flavus, A. parasiticus, A. nidulans, A. niger, Fusarium oxysporum, F. culmorum, Phytophthora nicotianae, Rhizoctonia solani, Pythium ultimum, Botrytis cinerea, Cercospora nicotianae, Thielaviopsis basicola, and Penicillium chrysogenum were obtained from the German Collection of Microorganisms and Cell Cultures
Following the immunization of mice with A. parasiticus cell wall fragments (CWFs), seven hybridoma clones producing Aspergillus-specific IgG antibodies were selected by enzymelinked immunosorbent assay (ELISA)

Summary

Introduction

The genus Aspergillus comprises 339 filamentous fungi that are ubiquitous in nature and have many potential applications in biotechnology, but some species pose a risk to human and animal health (Samson et al, 2014). The early detection of biomarkers elicited by invasive Aspergillus species is necessary to achieve effective antifungal therapy outcomes (Hedayati et al, 2007; Walsh et al, 2008). A. flavus and A. parasiticus infect plants, where they produce highly carcinogenic secondary metabolites known as aflatoxins, when they grow on oil-rich staple crops under field and storage conditions (Villers, 2014). These aflatoxins are stable during food processing, and contaminated food must be discarded causing significant economic losses amounting to billions of US$ in the US alone (Robens and Cardwell, 2003)

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