Innovative Assay Development for the Diagnosis of Invasive Aspergillosis

描述(由申请人提供)：侵袭性曲霉病(IA)是免疫功能低下患者中一种毁灭性的、经常是致命的感染，部分原因是早期检测工具不佳。目前，它导致52%的感染者死亡。对于曲霉病和其他侵袭性真菌疾病，迅速开展抗真菌治疗的能力对于积极的患者结果至关重要。然而，早期检测的复杂性在于发出疾病开始信号的抗原标记物水平较低，以及当前诊断化验的特异性较差。这项建议的目的是开发一个基于抗体的诊断平台，由新的单抗与表面增强拉曼光谱和金纳米颗粒夹心分析的超灵敏检测能力相结合。为了应对IA早期检测的挑战，已经组建了一个由犹他大学和北达科他州立大学的科学家组成的合作研究团队，成员包括在单抗开发和曲霉属筛选方面有专长的免疫学家；在基础真菌学和真菌检测开发和验证方面具有优势的医学微生物学家；专注于创造新颖的、超灵敏的和选择性诊断测试的分析化学家和纳米技术专家；以及熟练地设计和实施以患者为中心的研究的传染病临床医生。这项R21拨款申请将使用烟曲霉(A.fumigatus)--IA最常见的原因--作为原则证明目标来推进这一概念。这项R21资助申请的具体目标是：(1)为烟曲霉菌产生独特的、阶段特异性的生物标志物和相关的单抗。将产生针对烟曲霉菌细胞成分和分泌产物的鼠源单抗。(2)鉴定和优化基于表面增强拉曼散射(SERS)和单抗包被的金纳米颗粒标记的夹心免疫分析的多重单抗组合。(3)使用生物样品基质评估分析的性能。通过该项目的成功完成创建的检测战略将通过提高敏感性和特异性重新定义IA的诊断，允许在疾病的最早阶段进行治疗。 公共卫生相关性：侵袭性曲霉病(IA)是一种毁灭性的、经常致命的感染，发生在免疫功能低下和免疫抑制的患者中。不幸的是，对于IA和其他侵袭性真菌疾病，迅速开展抗真菌治疗的能力受到现有诊断工具的低临床敏感性、临床特异性和预后价值的影响。除非在早期阶段能够可靠地确定IA，否则伴随这种可怕疾病的发病率/死亡率将保持在令人无法接受的高水平。该项目的总体目标是开发一种快速、经济高效的实验室测试，作为自信的IA早期诊断的基石。

DESCRIPTION (provided by applicant): Invasive Aspergillosis (IA) is a devastating and frequently fatal infection in immunocompromised patients, in part because of poor early detection tools. It presently kills 52% of those infected. The ability to promptly launch antifungal therapy for Aspergillosis and other invasive fungal diseases is critical for positive patient outcomes. However, early detection is complicated by low levels of antigenic markers that signal disease onset and by the poor specificity of current diagnostic assays. The aim of this proposal is to develop an antibody- based diagnostics platform comprised of new monoclonal antibodies coupled with the ultrasensitive detection capabilities of surface enhanced Raman spectroscopy and gold nanoparticle sandwich assays. To address the challenges in the early detection of IA, a collaborative research team of scientists from the University of Utah and North Dakota State University has been assembled and includes immunologists with expertise in monoclonal antibody development and screening for Aspergillus spp; medical microbiologists with strengths in basic mycology, and in fungal assay development and validation; analytical chemists and nanotechnologists with a strong focus in the creation of novel, ultrasensitive and selective diagnostic tests; and infectious disease clinicians with proficiency in the design and implementation of patient-oriented research. This R21 grant application will advance this concept using Aspergillus fumigatus (A. fumigatus) - the most common cause of IA - as the proof-of-principle target. The specific aims for this R21 grant application are: (1) To generate unique, stage-specific biomarkers and associated monoclonal antibodies (mAbs) for A. fumigatus. Murine mAbs will be generated against cellular components and secreted products of A. fumigatus. (2) To identify and optimize multiplexed mAb combinations for a sandwich immunoassay based on surface enhanced Raman scattering (SERS), and mAb-coated gold nanoparticle labels. (3) To assess the performance of the assay using biological sample matrices. The detection strategy created through the successful completion of this project will redefine the diagnosis of IA through augmented sensitivity and specificity, permitting treatment at the earliest stages of disease. PUBLIC HEALTH RELEVANCE: Invasive aspergillosis (IA) is a devastating, frequently fatal infection in immunocompromised and immunosup- pressed patients. Unfortunately, the ability to promptly launch antifungal therapy for IA and other invasive fun- gal diseases is compromised by the poor clinically sensitivity, clinical specificity, and prognostic value of exist- ing diagnostic tools. Until IA can be reliably identified at early stages, the morbidity/mortality attendant with this dreadful disease will remain unacceptably high. The overall goal of this project is to develop a rapid, cost effec- tive laboratory test that will serve as a cornerstone for the confident early diagnosis of IA.