Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan

Abstract

A panel of specific monoclonal antibodies (mAbs) against synthetic pentasaccharide β-D-Galf-(1→5)-[β-D-Galf-(1→5)]3-α-D-Manp, structurally related to Aspergillus fumigatus galactomannan, was generated using mice immunized with synthetic pentasaccharide-BSA conjugate and by hybridoma technology. Two selected mAbs, 7B8 and 8G4, could bind with the initial pentasaccharide with affinity constants of approximately 5.3 nM and 6.4 nM, respectively, based on surface plasmon resonance-based biosensor assay. The glycoarray, built from a series of synthetic oligosaccharide derivatives representing different galactomannan fragments, demonstrated that mAb 8G4 could effectively recognize the parental pentasaccharide while mAb 7B8 recognizes its constituting trisaccharide parts. Immunofluorescence studies showed that both 7B8 and 8G4 could stain A. fumigatus cells in culture efficiently, but not the mutant strain lacking galactomannan. In addition, confocal microscopy demonstrated that Candida albicans, Bifidobacterium longum, Lactobacillus plantarum, and numerous gram-positive and gram-negative bacteria were not labeled by mAbs 7B8 and 8G4. The generated mAbs can be considered promising for the development of a new specific enzyme-linked assay for detection of A. fumigatus, which is highly demanded for medical and environmental controls.

Aspergillus fumigatus and Aspergillosis in 2019

“…Ex-panding the catalogue of epitopes recognized can explain the occurrence of some false-positive tests obtained with enzyme immunoassays (EIAs) using EB-A2 MAb (121,122). Recently, other anti-galactofuran antibodies that seem more specific to longer ␤1,5-galactofuranose oligosaccharides have been produced (123), but the performance of these two MAbs requires clinical validation.…”

Convergent Synthesis of Oligosaccharides Structurally Related to Galactan I and Galactan II of Klebsiella Pneumoniae and their Use in Screening of Antibody Specificity

“…Biotinylated glycoconjugates 1b – 6b were immobilized on the surface of streptavidin‐coated plates as described previously, to form glycoarray which was applied for screening of sera against K. pneumoniae O1 belonging to K‐serotypes 1, 2 and 16 . Anti‐sera were examined in 1:500 dilution (Figure ) as well as 1:250 and 1:1000 (see Supporting Information).…”

Reinvestigation of carbohydrate specificity of EB-A2 monoclonal antibody used in the immune detection of Aspergillus fumigatus galactomannan

“…Substitution of EB-A2 mAb in the immune assay with an antibody capable of recognizing a larger epitope should increase the specificity of the assay and will facilitate the decision for the initiation of an antifungal therapy. Synthetic immunogens which contain the oligosaccharide ligands of necessary length and structure can be regarded as promising instruments for obtaining of monoclonal antibodies with required epitope specificity and affinity to GM [32, 33] which may be better adapted for the conception of a more specific test in the serological diagnosis of invasive aspergillosis.…”