Protein Biomarkers for Invasive Aspergillosis Diagnostics

摘要 随着易感人群的增加，由烟曲霉引起的疾病负担已经 大幅增加。此外，由于诊断困难，实际受影响人数 人们可能被严重低估了。侵袭性曲霉病患者预后的改善 与早期诊断直接相关，强调需要更准确的诊断测试。在这些人中 对诊断化验提出的挑战包括：一、有机体的普遍存在，需要有能力 区分污染、感染和疾病；2.易感宿主的种类，在这些宿主中 疾病的发病机制可能会有所不同；疾病症候群的范围从过敏到 从曲菌瘤到侵袭性疾病的中介过程。目前，大部分精力都集中在诊断上 发展的重点是血清碳水化合物抗原的检测，这是基于聚合酶链式反应的真菌指标 用于检测抗真菌抗体反应的负荷法或免疫法。真菌蛋白质组分析 一般已局限于体外生长条件。此应用程序建议在 Marta FeldMesser博士(阿尔伯特·爱因斯坦医学院)与John Galgiani博士和Vicki Wysocki博士 (亚利桑那大学)在动物模型中识别与侵袭性疾病相关的特定蛋白质生物标记物 中性粒细胞减少和皮质类固醇治疗的小鼠，但在过敏性支气管肺模型中没有发现 曲霉病。真菌蛋白质组将被探测到允许更敏感的标记的范围 和疾病的特定标记物使用质谱学方法来识别生物标记物。 然后，重组蛋白将被用来制造可用于大量使用的单抗。 以光谱分析为基础的分析发展。提出了六个具体目标：1.鉴定烟曲霉菌蛋白 在侵袭性肺部疾病期间而不是在真菌性哮喘期间出现；2.表达和 三种侵袭性曲霉病候选生物标志物重组蛋白的纯化；3.制备单克隆 用于诊断分析的AIM 1中鉴定的真菌蛋白抗体；4.使用质谱仪 鉴定用于免疫分析的单抗；5.建立检测候选抗体的酶联免疫吸附试验 实验性侵袭性和过敏性曲霉病小鼠模型和临床标本中的生物标志物 其他NIH合同资源；以及6.开始将重组蛋白和单抗试剂迁移到 用于临床应用的多路和护理点平台。前两个目标将在以下期间实现 R21部分将在R33期间完成，其余4个将在R33期间完成。这个 这些研究的完成将为新的诊断方法提供潜在的灵活性 对特定临床环境的反应。

ABSTRACT 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.