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.
Highlights
Aspergillus fumigatus is the causative agent of a wide range of infections, the most common being allergic bronchopulmonary aspergillosis, local aspergillosis, chronic pulmonary aspergillosis, as well as invasive aspergilloses [1]
The present study reports two new galactomannan-recognizing monoclonal antibodies (mAbs) developed using Bovine serum albumin (BSA)-bound (GM-1-BSA) and biotinylated (GM-1-Biot) conjugates of synthetic pentasaccharide β-D-Galf-(1!5)-[β-DGalf-(1!5)]3-α-D-Manp derivative (GM-1) (Fig 1B)
8, and 12 weeks after the first immunization, mice sera were screened by indirect enzyme-linked immunosorbent assay (ELISA) to assess the level of anti- galactomannan IgG antibodies; biotinylated pentasaccharide GM-1-Biot was used as an antigen to exclude selection of anti-BSA mAbs
Summary
Aspergillus fumigatus is the causative agent of a wide range of infections, the most common being allergic bronchopulmonary aspergillosis, local (non-invasive) aspergillosis, chronic pulmonary aspergillosis, as well as invasive aspergilloses [1]. Invasive pulmonary aspergillosis has been a leading cause of infection-related deaths among immunocompromised patients [2]. V/ Tikunova); microorganisms used in this work were provided by the Collection of Extremophile Microorganisms and Type Cultures (developed within Project 0309-2017-0008 granted by the Russian Federal Agency of Scientific Organizations; http://fano.gov.ru); study of the recognition of cell cultures by monoclonal antibodies was supported by The French National Research Agency
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