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

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 (Gal f )-deficient mutant Δ glfA confirmed that Gal f residues are part of the epitope. Several lines of evidence strongly indicated that AP3 recognizes the Gal f residues of O -linked glycans on Aspergillus proteins. Glycoarray analysis revealed that AP3 recognizes oligo-[β-D-Gal f -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.

Characterization of a novel yeast phase-specific antigen expressed during in vitro thermal phase transition of Talaromyces marneffei

“…2b) similar to data previously described [7][8][9][10] . These results indicate that the target epitope of MAb 4D1 is shared by various forms of the glycoprotein according to previously described findings 12 . The GNA binding studies demonstrated that the majority of carbohydrate components in TM CYA consisted predominantly of mannose residues.…”

Importance of Candida Antigenic Factors: Structure-Driven Immunomodulation Properties of Synthetically Prepared Mannooligosaccharides in RAW264.7 Macrophages

“…Next, a novel tetrasaccharide construct consisting of β-1,2-mannopyranosyl trisaccharide and α-mannopyranoside was designed and suggested as a model of the C. albicans phosphodiester epitope (Dang et al, 2012). Glycoarrays formed by biotinylated oligosaccharides loaded on streptavidin-coated surfaces were previously shown to be indispensable instruments for the investigation of carbohydrate antigen recognition by immune cells (Komarova et al, 2015, 2018; Akhmatova et al, 2016; Paulovicova et al, 2016, 2017; Kurbatova et al, 2017; Argunov et al, 2019; Schubert et al, 2019).…”

Reinvestigation of Carbohydrate Specificity of EBCA-1 Monoclonal Antibody Used for the Detection of Candida Mannan

“…Modern methods of carbohydrate chemistry permit efficient and stereospecific assembling of complex oligosaccharide chains representing different parts of polysaccharides of the fungal cell wall [ 22 , 23 , 24 ]. The arrays of oligosaccharides (glycoarrays) were shown to be indispensable tools in the assessment of ligand specificity of anti-carbohydrate monoclonal antibodies [ 25 , 26 , 27 , 28 , 29 , 30 , 31 ] and of antibodies in the blood sera [ 26 , 32 , 33 , 34 ].…”