Selection and characterization of two monoclonal antibodies specific for the Aspergillus flavus major antigenic cell wall protein Aflmp1 |
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Institution: | 1. Department of Plant Molecular Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran;2. Department of Plant Viruses, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran;3. Department of Cell & Molecular Biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran;4. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074 Aachen, Germany |
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Abstract: | Aspergillus flavus is a major fungal pathogen of plants and an opportunistic pathogen of humans. In addition to the direct impact of infection, it produces immunosuppressive and carcinogenic aflatoxins. The early detection of A. flavus is therefore necessary to diagnose and monitor fungal infection, to prevent aflatoxin contamination of food and feed, and for effective antifungal therapy. Aspergillus-specific monoclonal antibodies (mAbs) are promising as diagnostic and therapeutic reagents for the tracking and treatment of Aspergillus infections, respectively. However, A. flavus has a complex cell wall composition and dynamic morphology, hindering the discovery of mAbs with well-characterized targets. Here we describe the generation and detailed characterization of mAb5.52 (IgG2aκ) and mAb17.15 (IgG1κ), which bind specifically to the highly immunogenic cell wall antigen A. flavus mannoprotein 1 (Aflmp1). Both mAbs were generated using hybridoma technology following the immunization of mice with a recombinant truncated version of Aflmp1 (ExD, including the homologous CR4 domain) produced in bacteria. We show that mAb5.52 and mAb17.15 bind specifically to A. flavus and A. parasiticus cell wall fragments (CWFs), with no cross-reaction to CWFs from other fungal pathogens. Immunofluorescence microscopy revealed that both mAbs bind to the surface of Aspergillus hyphae and that mAb17.15 also binds to spores. The epitope for both mAbs is localized within the CR4 region of the Aflmp1 protein. These Aspergillus-specific mAbs may be useful for the early detection of fungal infection in food/feed crops, for serodiagnosis in patients with invasive aspergillosis caused by A. flavus infection and for the development of antibody-expressing disease-resistant crops. |
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Keywords: | Aflatoxin Aspergillosis Detection assay Hybridoma technology Immunofluorescence microscopy Mannoproteins Plant pathogens Recombinant protein expression |
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