Novel mouse monoclonal antibodies specifically recognize <i>Aspergillus fumigatus</i> galactomannan

<div><p>A panel of specific monoclonal antibodies (mAbs) against synthetic pentasaccharide β-D-Gal<i>f</i>-(1→5)-[β-D-Gal<i>f</i>-(1→5)]₃-α-D-Man<i>p</i>, structurally related to <i>Aspergillus fumigatus</i> 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 <i>A</i>. <i>fumigatus</i> cells in culture efficiently, but not the mutant strain lacking galactomannan. In addition, confocal microscopy demonstrated that <i>Candida albicans</i>, <i>Bifidobacterium longum</i>, <i>Lactobacillus plantarum</i>, 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 <i>A</i>. <i>fumigatus</i>, which is highly demanded for medical and environmental controls.</p></div

Binding of fungal and bacterial cultures with mAbs 7B8 and 8G4.

<p>(A) Sandwich enzyme-linked immunosorbent assay (ELISA) with 7B8 mAb: the wells of microtiter plates were coated with 7B8 mAb and incubated with serial dilutions of microbial supernatants; ELISA was performed with horseradish peroxidase-conjugated 7B8 mAb. (B) Sandwich ELISA with 8G4 mAb: the wells of microtiter plates were coated with 8G4 mAb and incubated with serial dilutions of microbial supernatants; ELISA was performed with horseradish peroxidase-conjugated 8G4 mAb.</p

Investigation of oligosaccharide specificity of mAbs 7B8 and 8G4 using ELISA.

<p>(A) Composition of thematic glycoarray built using oligosaccharide ligands representing key structural elements of <i>A</i>. <i>fumigatus</i> galactomannan chain, and (B) assay for carbohydrate specificity of 7B8 and 8G4 mAbs.</p

Structure of <i>Aspergillus fumigatus</i> galactomannan and its synthetic analogs.

<p>(A) Structural fragments of <i>A</i>. <i>fumigatus</i> galactomannan (summarized from refs. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193938#pone.0193938.ref006" target="_blank">6</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193938#pone.0193938.ref008" target="_blank">8</a>]). (B) Pentasaccharide <b>GM-1</b> and its BSA <b>(GM-1-BSA)</b> and biotinylated <b>(GM-1-Biot)</b> conjugates used in mice immunization and mAb screening. The carbohydrate sequences are represented according to symbol carbohydrate nomenclature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193938#pone.0193938.ref026" target="_blank">26</a>].</p

Specific binding of mAbs 7B8 and 8G4 with <i>A</i>. <i>fumigatus</i>, <i>A</i>. <i>flavus</i>, and <i>C</i>. <i>albicans</i>.

<p>Cells were grown in Sabouraud broth, fixed, and incubated with mAbs 7B8 and 8G4. Binding of mAbs with fungal cells was detected with Alexa Fluor 488-conjugated anti-mouse IgG antibody staining in (A) confocal microscopy, and (B) DIC microscopy. Scale bar = 10 μm.</p