With the rise in susceptible individuals, the burden of disease due to Aspergillus fumigatus has increased substantially. In addition, because of the difficulties in diagnosis, the actual number of affected people likely has been severely underestimated. Improved outcome in patients with invasive aspergillosis has been directly linked to earlier diagnosis, underscoring the need for more accurate diagnostic tests. Among the challenges posed for diagnostic assays include: i. the ubiquity of the organism, necessitating the ability to distinguish between contamination, infection, and disease;ii. the variety of susceptible hosts, in whom the pathogenesis of disease may vary;iii. the spectrum of disease syndromes, which range from allergy-mediated processes to aspergilloma to invasive disease. At present, the majority of effort in diagnostic development has focused on serum detection of carbohydrate antigens, PCR-based indicators of fungal burden or immunoassays for detection of anti-fungal antibody responses. Fungal proteomic analyses generally have been limited to in vitro growth conditions. This application proposes a collaboration between Dr. Marta Feldmesser (Albert Einstein College of Medicine) with Drs. John Galgiani and Vicki Wysocki (University of Arizona) to identify specific protein biomarkers associated with invasive disease in animal models of neutropenic and corticosteroid-treated mice, but that are not found in a model of allergic bronchopulmonary aspergillosis. The fungal proteome will be probed for the range of markers that will allow for more sensitive and specific markers of disease using mass spectrometry approaches to biomarker identification. Recombinant proteins then will be used to create monoclonal antibodies that can be used for mass spectrometry-based assay development. Six specific aims are proposed: 1. To identify A. fumigatus proteins present specifically during invasive pulmonary disease and not during fungal asthma;2. to express and purify recombinant proteins of three candidate biomarkers of invasive aspergillosis;3. to make monoclonal antibodies to fungal proteins identified in Aim 1 for use in diagnostic assays;4. to use mass spectrometry to identify monoclonal antibodies for use in immunoassays;5. to develop a prototype ELISA to detect candidate biomarkers in experimental invasive and allergic aspergillosis murine models and in clinical specimens from other NIH contract resources;and 6. to begin to migrate the recombinant protein and MAb reagents to multiplex and point-of-care platforms for clinical applications. The first two Aims would be accomplished during the R21 portion of the award, while the remaining four would be done during the R33 period. The accomplishment of these studies would provide novel diagnostic methods with potential for flexibility in response to particular clinical settings.