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

开发抗沙门氏菌单链抗体作为血清诊断剂

• 批准号: 8302016
• 负责人: MICHAEL D GUNN
• 金额: $ 19.38万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8302016
• 关键词: Affinity; Africa; Antibiotic Therapy; Antibodies; Area; Asia; Bacteremia; Bacteriophages; Binding; Biological Assay; Biology; Blood; Bone Marrow; CD4 Positive T Lymphocytes; Case Fatality Rates; Cell Count; Cessation of life; Characteristics; Child; Collection; Detection; Development; Diagnosis; Diagnostic; Diagnostic Reagent; Diagnostic tests; Early Diagnosis; Early treatment; Engineering; Enteral; Enzyme-Linked Immunosorbent Assay; Epidemiology; Equipment and Supplies; Feces; Fingers; Goals; Grant; Human; Immune; Immunoassay; Immunoglobulin G; In Vitro; Infection; Institutes; Institution; Laboratories; Lateral; Lead; Libraries; Methods; Morbidity - disease rate; Mus; Mutate; Patients; Persons; Phage Display; Phase; Reagent; Recombinant Antibody; Recombinants; Reporting; Resources; Salmonella; Salmonella enterica; Salmonella paratyphi; Salmonella typhi; Sanguisorba; Screening procedure; Sensitivity and Specificity; Sepsis; Serotyping; Serum; Systemic infection; Technology; Testing; Training; Typhoid Fever; Urine; Variant; Water; Whole Blood; base; clinical care; cost; global health; improved; minimally invasive; mortality; novel; point of care; point-of-care diagnostics; prototype; rapid diagnosis; scale up

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. PUBLIC HEALTH RELEVANCE: The proposed studies are intended to lead directly to the development of a low-cost point of care diagnostic test for invasive S. enterica infection. The widespread availability of such a test would significantly improve global health by allowing the immediate diagnosis and rapid treatment of S. enterica infections.

描述（由申请方提供）：据估计，肠道沙门氏菌感染每年在全球造成约2500万例疾病和20万至25万例死亡。早期诊断和适当的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，据报告病死率为10-30%，这似乎是由于治疗机构的延误。目前诊断S.肠道感染包括血液、骨髓和粪便培养，但是昂贵、相对缓慢且通常在护理点不可用。在此，我们建议开发高敏感性和特异性的重组抗S。肠道单链抗体（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。肠特异性scFv，并开发这些试剂，以证明它们作为潜在诊断剂的实用性。在本项目的R21阶段，我们将识别S。通过筛选天然人和免疫小鼠scFv噬菌体文库来筛选肠道特异性噬菌体scFv克隆;将这些克隆表达和纯化为单体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。肠道抗体将构成本提案R33部分开发的特异性诊断测定的基础。R33部分将涉及三个方面。首先，我们将鉴定可用作ELISA测定的捕获和检测试剂的Ab对。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灵敏度和特异性。第三，我们将开发这些重组抗体作为使用侧流免疫测定格式的血清诊断。实现这些目标将导致开发一种能够检测S. enterica、S.伤寒沙门氏菌（S.小于100升全血中的副伤寒抗原。 公共卫生关系：拟议的研究旨在直接导致开发一种低成本的侵入性S。肠道感染这种测试的广泛可用性将通过允许S的即时诊断和快速治疗来显着改善全球健康。肠道感染