Development of Anti-Aspergillus fumigatus Monoclonal Antibodies
Biochemistry and Molecular Biology
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Aspergillus fumigatus is an opportunistic fungal pathogen responsible for causing the disease Invasive Aspergillosis (IA). It is difficult to diagnose the disease, given that patients suffering from IA exhibit symptoms similar to many other diseases. Due to rapid dissemination of fungal conidia to deep tissues during the course of infection, a rapid, affordable diagnostic test is needed to increase patient survival. Our lab has previously utilized the novel In Vivo Microbial Antigen Discovery (InMAD) technique to identify several candidate fungal proteins shed into the host as a result of infection. This project focused on the creation of monoclonal antibodies with specificity to these proteins for the ultimate use in lateral flow immunodiagnostics (LFI). Antibodies were generated through an anti-peptide approach and a genetic immunization scheme was evaluated as an alternative. Bioinformatics was used to ensure that all antibodies generated would have specificity for the A. fumigatus species and would be able to recognize surface-exposed regions of the protein. Murine immunizations led to the development of hybridoma cell lines for monoclonal antibodies production. Immunoassays and surface plasmon resonance (SPR) studies were carried out to assess the functional characteristics of the antibodies generated through the anti-peptide approach. The genetic workflow resulted in the development of DNA constructs suitable for murine immunization, as well as expressed protein fragments to be used as bi-weekly antigen boosts. These results suggest that the genetic immunization approach is possible and would serve as an alternative to the anti-peptide approach in developing monoclonal antibodies to be used in lateral flow immunodiagnostics.