Development of anti-Aspergillus single chain Abs as serum and imaging diagnostics

项目摘要 侵袭性曲霉病（IA）是最常见的丝状真菌感染， 免疫功能低下的患者。诊断为这种疾病的患者的死亡率范围 从70%到90%目前诊断IA的方法包括成像 技术和检测抗原、抗曲霉抗体或曲霉属 核酸不幸的是，目前的测试只有一个穷人到中等的准确性， 免疫功能低下患者侵袭性曲霉病的诊断。在这里，我们建议 研制高灵敏度、高特异性的重组抗曲霉菌单链抗体 抗体（scFv），并使用这些作为新的和准确的诊断测定的基础。 在这项资助的R21部分，我们的目标是产生高亲和力的单体或 多聚体曲霉特异性scFv，并开发这些试剂， 作为潜在诊断的实用性被证明。在目标1中，我们将确定曲霉菌- 通过筛选天然人scFv噬菌体文库获得特异性噬菌体scFv克隆。在目标2中， 将鉴定检测循环曲霉菌抗原的scFv。在目标3中，我们将表达 并将这些克隆纯化为单体scFv和各种多聚体构建体， 验证其约束力。在目标3中，我们将鉴定可用作 用于ELISA测定和诊断Ab阵列的捕获和检测试剂。在目标4中， 将产生放射性标记的曲霉菌结合试剂， 最高的亲和力、稳定性和目标密度。 R33部分将涉及三个方面。首先，我们会改善 通过产生和筛选我们的重组抗体， 这些抗体的随机突变变体。第二，我们将开发这些 重组抗体作为血清诊断，使用标准ELISA测定或 抗体阵列第三，我们将开发方法来检测曲霉菌感染，通过 使用重组抗体成像。

Project Summary Invasive aspergillosis (IA) is the most common filamentous fungal infection in immunocompromised patients. Mortality in patients diagnosed with this condition ranges from 70% to 90% at one year. Current methods to diagnose IA include imaging techniques and the detection of antigens, anti-Aspergillus antibodies, or Aspergillus nucleic acids. Unfortunately, current tests have only a poor to moderate accuracy for the diagnosis of invasive aspergillosis in immunocompromised patients. Here, we propose to develop highly sensitive and specific recombinant anti-aspergillus single-chain antibodies (scFvs) and using these as the basis for new and accurate diagnostic assays. In the R21 portion of this grant, our goal is to generate high affinity monomeric or multimeric aspergillus-specific scFvs and develop these reagents to the extent that their utility as potential diagnostics is demonstrated. In Aim 1, we will identify aspergillus- specific phage scFv clones by screening na¿ve human scFv phage libraries. In Aim 2, we will identify scFvs that detect circulating aspergillus antigens. In Aim 3, we will express and purify these clones as monomeric scFvs and various multimeric constructs and validate their binding activities. In Aim 3, we will identify scFv pairs that can be used as capture and detection reagents for ELISA assays and diagnostic Ab arrays. In Aim 4, we will generate radiolabeled aspergillus-binding reagents and identify those with the highest affinity, stability, and target density. The R33 portion of this grant will involve three aspects. First, we will improve the sensitivity and specificity of our recombinant antibodies by generating and screening randomly mutated variants of these antibodies. Second, we will development these recombinant antibodies as serum diagnostics using either standard ELISA assays or antibody arrays. Third, we will develop methods to detect Aspergillus infection via the use of recombinant antibody imaging.