This application is aimed at demonstrating the utility of viratrode technology for rapid detection of infectious disease specific protein markers in biologically relevant body fluids. For this purpose, Aspergillus fumigatus, a fungus that causes disease in patients with immunocompromised immune systems, has been selected as a model pathogen. Although there have been marked improvements in the early detection, diagnosis and treatment of Aspergillus, there is still a great need for the development of new technologies that will result in even better management of this disease. Therefore, a technology that reliably measures the presence of these markers in the blood or urine on a real-time basis could have a major impact on the early detection and diagnosis of infectious diseases, such as Aspergillosis, thus leading to improved outcomes in subsequent treatment.
The Specific Aims outlined in this Phase I SBIR AT application are aimed at demonstrating the ability of the viratrode to rapidly test a sample of blood, serum, urine or bronchoalveolar lavage fluid for the presence of an Aspergillus specific marker. In addition, pathogen detection is merely one potential use of the viratrode technology proposed here since the inherent design of the viratrode allows for it to be easily engineered to address a broad range of research and biomedical applications.
n this application, we intend to demonstrate the utility of viratrode technology for detecting molecules that are specific markers for pathogens in body fluids such as urine and serum. We have chosen to demonstrate this concept using Aspergillus fumigatus as a model pathogen because Aspergillosis poses a major health problem for patients with compromised immune systems. Recent work in the laboratory of Professor Margo Moore of Simon Fraser University has identified cyclic peptides of the siderophore family as promising targets for the early detection and diagnosis of aspergillosis. Thus, we propose to screen the phage libraries constructed by Professor Weiss to identify high affinity binders to N',N'',N'''- triacetylfusarinine C (TAF), the primary siderophore expressed by A. fumigatus upon infection. Phage displaying the high affinity TAF binders will be grown and used to prepare viratrodes in the Penner laboratory. The viratrodes will then be tested against samples obtained from the Invasive Aspergillosis consortium at the University of Texas Health Sciences Center San Antonio. If the proposed studies prove successful, viratrodes for other pathogens could be easily developed using the techniques described in this application. The primary goal of this project is thus focused on first establishing the ability of the viratrode to quantitatively detect TAF in biologically relevant fluids such as blood, serum, urine or bronchoalveolar lavage fluid collected from animals infected with Aspergillus fumigatus. Using established procedures, Professor Moore will purify TAF from cultures of Aspergillus fumigatus. The purified TAF will then be forwarded to Professor Weiss for selection of ligands that bind specifically to TAF by panning against several different phage libraries. Negative selections against other fungal extracts, other proteins in blood, serum, urine or bronchoalveolar lavage fluid will ensure specific binding to TAF. Phage expressing TAF specific ligands will be prepared and viratrodes constructed using techniques that have already been developed in the Penner laboratory. Detection of TAF by the viratrode will be assessed in biologically relevant fluids such as blood and serum, urine and bronchoalveolar lavage fluid obtained from the Invasive Aspergillosis consortium. Since early detection is of key importance for diagnosis of aspergillosis, samples will be taken from animals at various timepoints. Non-specific binding interactions will be assessed by screening against the Aspergillus negative viratrode. The best candidate viratrode will be selected based in maximal sensitivity and largest signal relative to the control electrode. TAF levels in these fluids will be validated in Dr. Moore's laboratory by HPLC.
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