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

描述（由申请人提供）：侵袭性曲霉病（IA）是免疫功能低下患者中最常见的丝状真菌感染。确诊为该病的患者一年内死亡率在70%至90%之间。目前诊断IA的方法包括成像技术和检测抗原、抗曲霉抗体或曲霉核酸。不幸的是，目前的检测方法对免疫功能低下患者侵袭性曲霉病的诊断准确度很低。在此，我们建议开发高敏感和特异性的重组抗曲霉单链抗体（scFvs），并将其作为新的准确诊断方法的基础。在这项资助的R21部分，我们的目标是产生高亲和力的单聚或多聚曲霉特异性scFvs，并开发这些试剂，以证明其作为潜在诊断的效用。在Aim 1中，我们将通过筛选5个人类scFv噬菌体文库来鉴定曲霉特异性噬菌体scFv克隆。在Aim 2中，我们将鉴定检测循环曲霉抗原的scFvs。在Aim 3中，我们将表达和纯化这些克隆为单体scFvs和各种多聚体构建体，并验证它们的结合活性。在Aim 3中，我们将鉴定可用于ELISA测定和诊断Ab阵列的捕获和检测试剂的scFv对。在Aim 4中，我们将生成放射性标记的曲霉结合试剂，并鉴定出具有最高亲和力、稳定性和靶密度的试剂。这个奖助金的R33部分将涉及三个方面。首先，我们将通过生成和筛选这些抗体的随机突变变体来提高重组抗体的敏感性和特异性。其次，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，我们将开发利用重组抗体成像检测曲霉感染的方法。

DESCRIPTION (provided by applicant): 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 develop 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.