Target discovery and immunoassay for 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发病和死亡的最严重原因之一。尽管进行了积极的抗真菌治疗，死亡率仍可能超过50%。治疗失败的一个主要原因是难以获得早期诊断，从而促进及时的抗真菌治疗。早期诊断可显著提高生存率。该项目的目标是开发一种检测血液或尿液中曲霉抗原的免疫测定方法，以促进IA的早期诊断。开发用于诊断的免疫测定法的困难在于确定体内产生的数百或数千种真菌抗原中哪一种会以足以检测的浓度出现在体液中。一种新的靶标发现方法，称为体内微生物抗原发现（InMAD），将用于确定免疫测定的候选蛋白抗原。InMAD策略是基于这样的假设，即感染烟曲霉的小鼠的血清或尿液将恰好含有那些将成为免疫测定目标的真菌蛋白。收集烟曲霉感染BALB/c小鼠的血清或尿液，过滤去除整个细胞/菌丝，但留下感染期间产生的可溶性抗原。这些可溶性抗原是免疫测定的潜在靶标。过滤后的样品将用于免疫无BALB/c小鼠。正常的BALB/c小鼠将真菌抗原视为外来物并产生抗体。从免疫小鼠中收集血清，利用真菌制备的一维和二维免疫印迹来鉴定抗体识别的抗原。这项研究将分两个阶段进行。第一个R21阶段将专注于目标发现。潜在的诊断靶点将使用InMAD方法确定，靶点将根据IA动物模型的血清或尿液分泌和曲霉菌特异性的确定进行验证。第二个R33阶段将是免疫测定的开发和评估。针对目标蛋白产生多克隆和单克隆抗体；将构建针对最有希望的蛋白质的免疫测定；免疫测定法将被评价为侵袭性曲霉病的诊断手段。最终的产品将是一个免疫分析，确定两种或更多不同的曲霉特异性抗原的存在。InMAD方法在兔热病、类melidosis和回归热中发现靶点的概念证明已经掌握，其中在感染期间已确定进入血清的候选蛋白质和多糖。

DESCRIPTION (provided by applicant): Invasive aspergillosis (IA) is one of the most serious causes of morbidity and mortality among immune compromised patients. Mortality may exceed 50% despite aggressive antifungal therapy. A major cause of treatment failure is the difficulty in obtaining an early diagnosis that would facilitate timely antifungal therapy. Early diagnosis can markedly improve survival. The goal of this project is development of an immunoassay for detection of Aspergillus antigens in blood or urine to facilitate early diagnosis of IA. The difficulty in development of immunoassays for diagnosis is a determination of which of the potentially hundreds or thousands of fungal antigens produced in vivo will show up in body fluids in concentrations sufficient for detection. A novel approach to target discovery, termed In vivo Microbial Antigen Discovery (InMAD), will be used to identify candidate protein antigens for immunoassay. The InMAD strategy is based on the hypothesis that serum or urine from mice infected with Aspergillus fumigatus will contain precisely those fungal proteins that would be targets for immunoassay. Serum or urine from A. fumigatus-infected BALB/c mice will be collected and filtered to remove whole cells/hyphae but leave behind soluble antigens generated during infection. These soluble antigens are potential targets for immunoassay. The filtered samples will be used to immunize na¿ve BALB/c mice. Na¿ve BALB/c mice will see the fungal antigens as foreign and will make antibodies. Serum will be collected from the immunized mice and used to identify antigens recognized by the antibodies by use of one and two dimensional immunoblots prepared from the fungus. The study will occur in two phases. The first R21 phase will focus on target discovery. Potential diagnostic targets will be identified using the InMAD approach and targets will be validated on the basis of secretion into serum or urine in animal models of IA and a determination of specificity for Aspergillus spp. The second R33 phase will be immunoassay development and evaluation. Polyclonal and monoclonal antibodies will be produced to target proteins; immunoassays will be constructed that target the most promising proteins; and immunoassays will be evaluated as a means for diagnosis of invasive aspergillosis. The ultimate product will be an immunoassay that identifies the presence of two or more distinct Aspergillus- specific antigens. Proof of concept for the InMAD approach to target discovery is already in hand for tularemia, melioidosis and relapsing fever where candidate proteins and polysaccharides that are shed into serum during infection have been identified.