Target discovery and immunoassay for diagnosis of invasive aspergillosis

侵袭性曲霉病（IA）是免疫系统中最严重的致病和致死原因之一， 妥协的病人尽管进行了积极的抗真菌治疗，死亡率仍可能超过50%。的主要原因 治疗失败是难以获得有助于及时抗真菌治疗的早期诊断。 早期诊断可显著提高生存率。该项目的目标是开发一种免疫测定法， 检测血液或尿液中的曲霉菌抗原，以促进IA的早期诊断。的困难 用于诊断的免疫测定法的发展是确定潜在的数百种或更多种免疫测定法中的哪一种。 体内产生的数千种真菌抗原将以足够的浓度出现在体液中， 侦测一种新的靶点发现方法，称为体内微生物抗原发现（InMAD），将在 用于鉴定免疫测定的候选蛋白抗原。InMAD战略基于以下假设： 感染烟曲霉菌的小鼠的血清或尿液中恰恰含有这些真菌蛋白 这将是免疫分析的目标。血清或尿液来自A.感染烟曲霉的BALB/c小鼠将被 收集并过滤以除去全细胞/菌丝，但留下在培养期间产生的可溶性抗原。 感染这些可溶性抗原是免疫测定的潜在靶标。过滤后的样本将用于 免疫BALB/c小鼠。幼稚的BALB/c小鼠会将真菌抗原视为外来抗原， 抗体的将从免疫的小鼠收集血清并用于鉴定由免疫原性抗体识别的抗原。 通过使用从真菌制备的一维和二维免疫印迹来检测抗体。 研究将分两个阶段进行。R21的第一阶段将侧重于目标发现。潜在诊断 将使用InMAD方法鉴定靶点，并根据分泌到 IA动物模型中的血清或尿液，并测定对曲霉属的特异性。第二台R33 阶段将是免疫测定开发和评价。多克隆和单克隆抗体将被 将构建针对最有希望的蛋白质的免疫测定;以及 免疫测定将作为侵袭性曲霉病的诊断手段进行评价。 最终产品将是一种免疫测定法，可识别两种或多种不同曲霉菌的存在- 特异性抗原InMAD方法发现目标的概念验证已经在土拉菌病的研究中， 类鼻疽和回归热，其中候选蛋白质和多糖在治疗过程中脱落到血清中， 感染已被确认。

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.