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

描述（由申请人提供）：据估计，肠炎沙门氏菌感染每年在全球造成约2500万人患病，20万至25万人死亡。早期诊断和适当治疗对于这种感染的最佳管理至关重要，特别是在儿童中。在非洲和亚洲的一些地区，已报告的病死率为10-30%，这似乎是由于治疗机构延误造成的。目前诊断肠球菌感染的方法包括血液培养、骨髓培养和粪便培养，但这些方法昂贵、速度相对较慢，而且在医疗点一般无法获得。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（scFvs），并将其作为新的准确诊断分析的基础。在该资助的R21部分，我们的目标是产生高亲和力的单聚或多聚肠链球菌特异性scFvs，并开发这些试剂，以证明其作为潜在诊断的效用。在该项目的R21阶段，我们将通过筛选幼稚的人和免疫小鼠scFv噬菌体文库来鉴定肠链球菌特异性噬菌体scFv克隆；将这些克隆表达纯化为单体scFvs，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anti-S。enterica Abs将成为本提案R33部分开发的特定诊断检测的基础。这个奖助金的R33部分将涉及三个方面。首先，我们将确定可用于ELISA测定的捕获和检测试剂的Ab对。其次，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。第三，我们将开发这些重组抗体作为使用侧流免疫分析格式的血清诊断。实现这些目标将导致开发一种原型横向流动试验，能够检测不到100 μ l全血中的肠沙门氏菌、伤寒沙门氏菌和副伤寒沙门氏菌Ags。

DESCRIPTION (provided by applicant): 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 naive 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 ¿l of whole blood.