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

描述(申请人提供)：侵袭性曲霉病(IA)是免疫功能低下患者最常见的丝状真菌感染。被诊断为这种情况的患者一年的死亡率从70%到90%不等。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，目前的检测对免疫受损患者的侵袭性曲霉病的诊断只有很差到中等的准确性。在这里，我们建议开发高度敏感和特异的重组抗曲霉菌单链抗体(ScFv)，并将其作为新的准确诊断分析的基础。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多聚曲霉特异性单链抗体，并开发这些试剂，以证明它们作为潜在诊断的有效性。在目标1中，我们将通过筛选原始人源ScFv噬菌体文库来鉴定曲霉菌特异性单链抗体克隆。在目标2中，我们将鉴定检测循环曲霉抗原的单链抗体。在目标3中，我们将以单体单链抗体和各种多聚体结构表达和纯化这些克隆，并验证它们的结合活性。在目标3中，我们将确定可用作捕获和检测试剂的单链抗体对，用于ELISA检测和诊断抗体阵列。在目标4中，我们将生产放射性标记的曲霉结合试剂，并鉴定那些具有最高亲和力、稳定性和靶向密度的试剂。这笔赠款的33卢比部分将涉及三个方面。首先，我们将通过产生和筛选这些抗体的随机突变变体来提高我们的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的酶联免疫吸附试验或抗体阵列。第三，我们将开发通过使用重组抗体成像来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.