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的一种破坏性且经常致命的感染，部分原因是早期检测工具不完善。目前有52%的感染者死亡。及时启动抗真菌治疗曲霉病和其他侵袭性真菌疾病的能力对患者的积极结果至关重要。然而，早期检测由于表明疾病发病的抗原标记物水平低以及当前诊断分析的特异性差而变得复杂。本提案的目的是开发一种基于抗体的诊断平台，该平台由新的单克隆抗体结合表面增强拉曼光谱和金纳米颗粒夹心测定的超灵敏检测能力组成。为了解决IA早期检测的挑战，来自犹他大学和北达科他州立大学的科学家组成了一个合作研究小组，其中包括具有单克隆抗体开发和筛选曲霉菌的专业免疫学家；在基础真菌学和真菌分析开发和验证方面具有优势的医学微生物学家；分析化学家和纳米技术专家，专注于创造新颖、超灵敏和选择性的诊断测试；与传染病临床医生熟练设计和实施以病人为导向的研究。这项R21资助申请将利用烟曲霉（IA的最常见原因）作为原理证明目标来推进这一概念。该R21资助申请的具体目标是：(1)为烟曲霉产生独特的、阶段特异性生物标志物和相关单克隆抗体（mab）。针对烟曲霉的细胞成分和分泌产物生成小鼠单克隆抗体。(2)基于表面增强拉曼散射（SERS）和单抗包覆金纳米颗粒标记的三明治免疫分析，鉴定和优化多重单抗组合。(3)评估使用生物样品基质的检测性能。通过成功完成该项目创建的检测策略将通过增强的敏感性和特异性重新定义IA的诊断，允许在疾病的早期阶段进行治疗。

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.