Development of anti-Salmonella Single-chain Abs as serum diagnostics

项目摘要 感染肠道沙门氏菌估计导致约2500万人患病，20万人至 全球每年有25万人死亡。早期诊断和适当的治疗是最佳治疗方案的关键 对这种感染的管理，特别是在儿童中。在非洲和亚洲的一些地区， 已报告10-30%，这似乎是由于延迟的治疗机构造成的。当前 诊断肠链球菌感染的方法包括血液、骨髓和粪便培养，但 价格昂贵、速度相对较慢，并且在护理时通常不可用。 在这里，我们建议开发高度敏感和特异的重组抗S。肠系单链 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多聚体肠杆菌- 并开发这些试剂，以使其作为潜在诊断的效用 演示了。在这个项目的R21阶段，我们将通过以下方式识别肠杆菌特异性噬菌体单链抗体克隆 筛选原始人和免疫鼠单链抗体噬菌体文库；表达和纯化这些克隆 单链抗体并验证其结合活性；将最有希望的克隆转化为完整的免疫球蛋白 并验证其作为诊断试剂的实用性。这些反S。Enterica Abs将成为 具体的诊断分析将在本提案的R33部分中开发。 这笔赠款的33卢比部分将涉及三个方面。首先，我们将识别可用作 用于酶联免疫吸附试验的捕获和检测试剂。二是提高诊断的敏感性和特异性。 我们的重组抗体是通过产生和筛选这些抗体的随机突变变体来实现的。 第三，我们将开发这些重组抗体作为血清诊断使用侧向流动免疫分析。 格式化。实现这些目标将导致开发一种能够 在少于100微升的全血中检测肠杆菌、伤寒沙门氏菌和副伤寒沙门氏菌AGS。

Project Summary Infection with Salmonella enterica is estimated to cause approximately 25 million illnesses and 200,000 to 250,000 deaths per year globally. Early diagnosis and appropriate treatment are essential for optimal management of this infection, especially in children. In some areas of Africa and Asia, case fatality rates of 10-30% have been reported, which appears to be due to the delayed institution of treatment. Current methods to diagnose S. enterica infection include culture of blood, bone marrow, and stool, but are expensive, relatively slow, and not generally available at the point of care. Here, we propose to develop highly sensitive and specific recombinant anti-S. enterica 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 S. enterica- specific scFvs and develop these reagents to the extent that their utility as potential diagnostics is demonstrated. In the R21 phase of this project, we will identify S. enterica-specific phage scFv clones by screening na¿ve human and immune mouse scFv phage libraries; express and purify these clones as monomeric scFvs and validate their binding activities; and convert the most promising clones to intact IgG molecules and validate their utility as diagnostic reagents. These anti-S. enterica Abs will form the basis of specific diagnostic assays to be developed in the R33 portion of this proposal. The R33 portion of this grant will involve three aspects. First, we will identify Ab pairs that can be used as capture and detection reagents for ELISA assays. Second, we will improve the sensitivity and specificity of our recombinant antibodies by generating and screening randomly mutated variants of these antibodies. Third, we will develop these recombinant antibodies as a serum diagnostic using a lateral flow immunoassay format. Accomplishing these aims will result in the development of a prototype lateral flow assay capable of detecting S. enterica, S. typhi, and S. paratyphi Ags in less than 100 ul of whole blood.