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

项目摘要 据估计，肠道沙门氏菌感染导致约2500万人患病，20万人死亡。 全球每年有25万人死亡。早期诊断和适当的治疗是至关重要的， 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， 10-30%已报告，这似乎是由于治疗机构延迟。电流 诊断S.肠道感染包括血液、骨髓和粪便培养，但 昂贵、相对缓慢并且在护理点通常不可用。 在此，我们建议开发高敏感性和特异性的重组抗S。肠单链 抗体（scFv），并使用这些作为新的和准确的诊断测定的基础。 在R21部分，我们的目标是产生高亲和力的单体或多聚体S。肠的，肠的 特异性scFv，并开发这些试剂，以使其作为潜在诊断剂的实用性 演示。在本项目的R21阶段，我们将识别S。肠道特异性噬菌体scFv克隆 筛选天然人和免疫小鼠scFv噬菌体文库;表达和纯化这些克隆， 单体scFv并验证其结合活性;并将最有希望的克隆转化为完整的IgG 分子并验证其作为诊断试剂的效用。这些反S。肠道抗体将构成基础， 在本提案的R33部分中开发的特定诊断测定。 R33部分将涉及三个方面。首先，我们将确定可以用作 用于ELISA测定的捕获和检测试剂。其次，我们将提高灵敏度和特异性， 通过产生和筛选这些抗体的随机突变变体来获得我们的重组抗体。 第三，我们将开发这些重组抗体作为使用侧流免疫测定的血清诊断 格式.实现这些目标将导致开发出一种原型侧流测定法，其能够 检测S. enterica、S.伤寒沙门氏菌（S.小于100 μ l全血中的副伤寒抗原。

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.