Development of a fluorescence-based assay to detect fungus-derived proteolytic ac

开发基于荧光的检测方法来检测真菌来源的蛋白水解酶

• 批准号: 7779445
• 负责人: Amit Galande
• 金额: $ 26.16万
• 依托单位: SRI INTERNATIONAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7779445
• 关键词: Animal Model; Animals; Antibodies; Antifungal Agents; Antifungal Therapy; Antigens; Aspergillosis; Aspergillus; Aspergillus fumigatus; Biological; Biological Assay; Body Fluids; Breathing; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Buffers; Cell Wall; Characteristics; Clinical; Complex; Contracts; Detection; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Disease; Early Diagnosis; Environment; Enzymes; Fluorescence; Foundations; Fungal DNA; Genome; Goals; Human; Immunocompromised Host; In Vitro; Infection; Laboratory Finding; Libraries; Life; Lung; Measurable; Methodology; Methods; Molds; Molecular Target; Monitor; Nutrient; Organism; Outcome; Patients; Peptide Hydrolases; Phase; Polysaccharides; Population; Property; Reaction; Reproduction spores; Sampling; Serum; Source; Specimen; Symptoms; Technology; Testing; Tissues; Translating; Validation; Virulence; Work; assay development; base; combinatorial; diagnostic accuracy; effective therapy; fluorophore; fungus; immunosuppressed; improved; in vivo; mortality; novel; novel diagnostics; outcome forecast; pathogen; protein aminoacid sequence; rapid diagnosis; success; tool

DESCRIPTION (provided by applicant): Aspergillus fumigatus is the most prevalent airborne fungal pathogen, causing severe and usually fatal invasive infections in immunocompromised hosts. Success of anti-fungal therapy relies heavily on accuracy of diagnosis, but rapid and reliable detection of A. fumigatus remains difficult owing to the non-specific clinical symptoms and lack of distinct molecular targets. This proposal aims to utilize thermostable proteases secreted by A. fumigatus into the host environment as a target for the development of a novel fluorescence-based diagnostic assay for invasive aspergillosis. The applicant hypothesizes that fungal proteolytic activity can be detected in serum and BAL samples during invasive aspergillosis using a panel of Internally Quenched Fluorogenic Probes (IQFPs). Aim 1 of the R21 phase will use a commercially available library of 360 IQFPs to optimize assay conditions for maximal detection of fungal proteolytic activity secreted in vitro. These conditions will then be used to test the feasibility of the assay for the detection of A. fumigatus in serum and BAL samples from animal models of infection (Source: IAAM contract). In Aim 2, a panel of eight IQFPs with most promising diagnostic properties will be selected for targeted optimization in order to maximize sensitivity and selectivity. The R33 phase we will then evaluate the efficacy of this IQFP-based diagnostic platform for the detection of A. fumigatus proteases in clinical samples from human patients (Source: AsTeC contract). In addition, a comprehensive combinatorial library of 640,000 IQFPs will be assembled in order to expand the diagnostic applications to include the monitoring of antifungal therapy and the detection of different pathogenic moulds.

描述(由申请人提供)：烟曲霉是最常见的空气传播真菌病原体，在免疫功能低下的宿主中会引起严重的、通常是致命的侵袭性感染。抗真菌治疗的成功在很大程度上取决于诊断的准确性，但由于临床症状的非特异性和缺乏明确的分子靶点，快速、可靠地检测烟曲霉菌仍然是困难的。本研究旨在利用烟曲霉菌分泌的耐热蛋白水解酶作为靶标，发展一种新的基于荧光的侵袭性曲霉病诊断方法。申请人假设，在侵袭性曲霉病期间，可以使用一组内部淬灭的荧光探针(IQFP)在血清和BAL样本中检测到真菌蛋白分解活性。R21阶段的目标1将使用商业上可用的360个IQFP文库来优化测试条件，以最大限度地检测体外分泌的真菌蛋白分解活性。这些条件将被用来测试在动物感染模型的血清和BAL样本中检测烟曲霉菌的可行性(来源：IAAM合同)。在目标2中，将选择具有最有前景的诊断特性的八个IQFP小组进行有针对性的优化，以最大限度地提高灵敏度和选择性。在R33阶段，我们将评估这个基于IQFP的诊断平台在检测人类患者临床样本中烟曲霉蛋白酶的有效性(来源：ASTEC合同)。此外，还将组装一个由64万个IQFP组成的综合组合库，以扩大诊断应用，包括监测抗真菌治疗和检测不同的致病霉菌。