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

项目摘要 侵袭性曲霉病(IA)是我国最常见的丝状真菌感染 免疫功能受损的病人。被诊断为这种情况的患者的死亡率范围 一年内从70%增加到90%。目前诊断IA的方法包括成像 技术和抗原、抗曲霉抗体或曲霉的检测 核酸。不幸的是，目前的测试只有很差到中等的准确性 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、高特异性的重组抗曲霉单链抗体 抗体(ScFv)，并将其作为新的和准确的诊断分析的基础。 在这笔赠款的R21部分，我们的目标是产生高亲和力的单体或 曲霉菌特异性多聚体单链抗体，并将这些试剂开发到其 演示了作为潜在诊断的实用程序。在目标1中，我们将鉴定曲霉菌- 通过筛选人源ScFv噬菌体文库获得特异性ScFv噬菌体克隆。在目标2中，我们 将识别检测循环曲霉菌抗原的单链抗体。在目标3中，我们将表达 并将这些克隆纯化为单体单链抗体和各种多聚体构建体 验证它们的绑定活动。在目标3中，我们将识别可用作 用于酶联免疫吸附试验和诊断抗体阵列的捕获和检测试剂。在目标4中，我们 将产生放射性标记的曲霉结合试剂，并将其与 最高的亲和力、稳定性和目标密度。 这笔赠款的33卢比部分将涉及三个方面。首先，我们将完善 制备和筛选重组抗体的敏感性和特异性 这些抗体的随机突变变体。第二，我们将发展这些 重组抗体作为血清诊断的标准酶联免疫吸附试验或 抗体阵列。第三，我们将开发检测曲霉感染的方法，通过 利用重组抗体进行成像。

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.