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.

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Great progresses have been made in the recent years in the detection of circulating galactofuranose-bearing molecules for the diagnosis of aspergillosis. However, the test used in the clinical practice is hampered by the occurrence of false positives. A glycoarray with dozens of oligosaccharides structurally related to the Aspergillus fumigatus galactomannan has allowed us to reinvestigate the carbohydrate specificity of the EB-A2 monoclonal antibody used in the PlateliaTM Aspergillus sandwich immune assay. We have now demonstrated that the mAb can recognize shorter oligosaccharides than the previously reported tetrasaccharide Galf-β-(1→5)-Galf-β-(1→5)-Galf-β-(1→5)-Galf-β and oligosaccharides which contains alternating β-(1→5)/β-(1→6)-linkages. This result could explain the occurrence of false-positive signals due to the presence of the abovementioned epitopes not only in A. fumigatus galactomannan but also in other bacteria and fungi.

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