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

描述(由申请人提供)：感染肠道沙门氏菌估计每年导致全球约2500万人患病和20万至25万人死亡。早期诊断和适当治疗对于最有效地管理这种感染至关重要，尤其是在儿童中。在非洲和亚洲的一些地区，报告的病死率为10%-30%，这似乎是由于延误了治疗机构。目前诊断肠链球菌感染的方法包括血液、骨髓和粪便培养，但成本较高，相对较慢，而且在护理点通常不能获得。在这里，我们建议开发高度敏感和特异的重组抗S。肠道单链抗体(ScFv)，并利用这些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链抗体或多聚体肠杆菌特异性单链抗体，并开发这些试剂，以证明它们作为潜在诊断的有效性。在本项目的R21阶段，我们将通过筛选原始的人和免疫小鼠单链抗体噬菌体文库来鉴定肠杆菌特异性单链抗体克隆；表达和纯化这些克隆作为单体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G分子，并验证其作为诊断试剂的有效性。这些反S。肠道抗体将构成本提案R33部分将要开发的特定诊断分析的基础。这笔赠款的33卢比部分将涉及三个方面。首先，我们将确定可用作捕获和检测试剂的抗体对用于酶联免疫吸附试验。其次，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。第三，我们将开发这些重组抗体作为血清诊断使用横向流动免疫分析格式。实现这些目标将导致开发一种能够检测不到100%L全血中的肠杆菌、伤寒沙门菌和副伤寒沙门氏菌AGS的原型侧向流动试验。

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.