Target discovery and immunoassay for diagnosis of invasive aspergillosis

侵袭性曲霉病(IA)是免疫性疾病中发病率和死亡率最严重的原因之一。 有危险的病人。尽管进行了积极的抗真菌治疗，死亡率仍可能超过50%。一个主要原因是 治疗失败是获得早期诊断的困难，这将有助于及时进行抗真菌治疗。 早期诊断可明显提高存活率。该项目的目标是开发一种免疫分析方法，用于 检测血液或尿液中的曲霉菌抗原有助于IA的早期诊断。的困难在于 用于诊断的免疫分析的发展是确定数百种潜在的或 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。一种新的靶标发现方法，称为体内微生物抗原发现(InMAD)，将是 用于鉴定免疫分析的候选蛋白质抗原。InMAD策略基于以下假设 被烟曲霉菌感染的小鼠的血清或尿液中就含有这些真菌蛋白 这将是免疫分析的目标。感染烟曲霉菌的BALB/c小鼠的血清或尿液将被 收集和过滤以去除整个细胞/菌丝，但留下在 感染。这些可溶性抗原是免疫分析的潜在靶点。过滤后的样品将用于 免疫新生BALB/c小鼠。新的BALB/c小鼠将把真菌抗原视为外来的，并将使 抗体。将从免疫的小鼠中收集血清，并用于识别由 通过使用从真菌制备的一维和二维免疫印迹来获得抗体。 研究将分两个阶段进行。第一个R21阶段将专注于目标发现。潜在诊断 将使用InMAD方法确定目标，并将根据分泌到 IA动物模型的血清或尿液及曲霉菌的特异性测定。第二辆R33 阶段将是免疫分析的发展和评估阶段。多克隆和单克隆抗体将是 针对目标蛋白质生产；将构建针对最有希望的蛋白质的免疫分析；以及 免疫分析将被评估为诊断侵袭性曲霉病的一种手段。 最终的产品将是一种免疫分析，它可以识别两种或两种以上不同曲霉的存在- 特定的抗原。对于图拉热症，InMAD方法发现目标的概念证明已经在手中， 类鼻疽和复发性发热，这些候选蛋白质和多糖在 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.