Enrichment and validation of urine and serum-specific antigens from acute Lyme disease patient samples

急性莱姆病患者样本中尿液和血清特异性抗原的富集和验证

• 批准号: 10539304
• 负责人: David P AuCoin
• 金额: $ 18.46万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-10 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10539304
• 关键词: Acute; Animals; Antibodies; Antibody Response; Antigen Presentation; Antigens; Arthritis; Bacterial Antigens; Biological; Biological Assay; Borrelia burgdorferi; Carditis; Chronic; Clinic; Clinical; Clinical Sensitivity; Collaborations; Collection; Complex; Data; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Early Diagnosis; Early treatment; Ensure; Enzyme-Linked Immunosorbent Assay; Future; Healthcare; Human; Immunoassay; Immunoprecipitation; Impairment; Infection; Infectious Agent; Laboratories; Lyme Disease; Lyme disease diagnosis; Macaca; Magnetism; Mass Spectrum Analysis; Methods; Modeling; Neurologic; Nevada; Order Spirochaetales; Outcome Study; Patient Agents; Patient-Focused Outcomes; Patients; Procedures; Proteins; Protocols documentation; Public Health; Publishing; Rapid diagnostics; Recording of previous events; Research; Sampling; Serology; Serology test; Serum; Source; Techniques; Testing; Ticks; Time; United States; Universities; Urine; Validation; Variant; Vector-transmitted infectious disease; Western Blotting; antigen detection; antigen diagnostic; care burden; design; detection assay; erythema migrans; experience; improved; innovation; medical schools; member; prevent; tick-borne

ABSTRACT Lyme borreliosis is the leading vector-borne disease in the United States. Early diagnosis and treatment of Lyme disease is critical to prevent the development of severe complications such as extensive arthritis, chronic neurological impairments, and carditis. A critical barrier in the clinic is the inability to diagnose Lyme disease early during infection in patients that do not develop a distinct erythema migrans. The only FDA cleared tests for Lyme disease are two-tier serologic assays that are laborious to perform. More importantly, such assays are unable to diagnose Lyme disease in its early stages due to the time that is required for a patient to develop an antibody response to the infecting Borrelia burgdorferi (Bb), the causative agent of Lyme disease. In this proposal, our objective is to identify and validate urine and serum-specific antigens in samples collected from patients with Lyme disease. Validated antigens will be used, as part of a future proposal, to develop Bb antigen detection assays for the early diagnosis of Lyme disease. This is a challenging research endeavor that we believe will require an unwavering focus on the following two hypotheses: First, development of a diagnostic test detecting only one Bb antigen will not result in a clinically-useful test for early Lyme disease. We strongly believe that targeting multiple antigens, as opposed to a single antigen, will be required for the development of a highly-sensitive assay. During our preliminary studies we optimized methods to discover antigens that are present within samples collected from Lyme patients as well as a tick-borne macaque model of Lyme disease. Six candidate Bb antigens were identified within serum; these antigens will be considered for validation in patient samples through this R21 proposal. To demonstrate that the use of the DIA-MS approach, described in this proposal, will result in the consistent detection of potential antigens results from a preliminary study in which 5 acute patient serum samples were analyzed using the proposed method. The analysis found that 243 proteins were detected in all 5 samples, confirming the reliability of the approach. The reliable detection of Bb-specific antigens from biological samples is a significant diagnostic breakthrough, however, detection of multiple Lyme antigens will be essential to achieve high clinical sensitivity. Second, a diagnostic test for early Lyme disease will require patient sample enrichment prior to downstream assays. This has become clear from our preliminary studies with Lyme samples, as well as our studies to detect other infectious agents from patient samples. The development of sample enrichment protocols that require minimal processing will ensure that enrichment is compatible with the workflow of a clinical laboratory. A collaboration with John Hopkins Lyme Disease Research Center will allow for the inclusion of paired human urine and serum samples in the discovery and validation stages of the project. Ultimately, simplifying the diagnosis of Lyme disease will allow for an earlier diagnosis, improved patient outcome, and an easement of Lyme-related healthcare burdens.

抽象的 莱姆毛毛虫病是美国领先的载体传播疾病。早期诊断和治疗 莱姆病对于防止发生严重并发症的发展至关重要，例如广泛的关节炎，慢性 神经障碍和心脏炎。诊所的关键障碍是无法诊断莱姆病 在感染期间，患者没有出现明显的红斑迁移。 FDA唯一清除测试 对于莱姆病是两层血清学测定，努力执行。更重要的是，这样的测定 由于患者发育所需的时间，无法在早期诊断莱姆病诊断 对莱姆病的病因的伯氏伯氏菌（BB）的抗体反应。在这个 提案，我们的目标是在样品中识别和验证尿液和血清特异性抗原 从莱姆病患者中收集。经过验证的抗原将被用作未来建议的一部分 开发BB抗原检测测定法，以早期诊断莱姆病。这是一项具有挑战性的研究 我们认为，努力将需要坚定不移地关注以下两个假设：首先，发展 仅检测一个BB抗原的诊断测试不会导致早期莱姆的临床用途测试 疾病。我们坚信，将需要靶向多种抗原，而不是单个抗原 用于开发高度敏感的测定法。在我们的初步研究中，我们优化了 发现从莱姆患者收集的样品中存在的抗原以及tick传播的 莱姆病的猕猴模型。在血清中鉴定出六个候选BB抗原。这些抗原会 通过此R21提案在患者样品中进行验证。证明使用 该提案中描述的DIA-MS方法将导致对潜在抗原结果的一致检测 从一项初步研究中，使用该方法分析了5个急性患者血清样品。 分析发现，在所有5个样品中都检测到243种蛋白质，从而确认了该方法的可靠性。 从生物样品中对BB特异性抗原的可靠检测是一个显着的诊断突破， 但是，多种莱​​姆抗原的检测对于达到高临床敏感性至关重要。第二，a 早期莱姆病的诊断测试将需要在下游测定之前患者样品富集。这 从我们对莱姆样本的初步研究中可以清楚地看出，我们的研究以检测其他 来自患者样品的传染剂。需要最小的样品富集协议的开发 处理将确保富集与临床实验室的工作流程兼容。合作 使用约翰·霍普金斯莱姆病研究中心，将允许配对人类尿液和血清 在项目的发现和验证阶段中的样本。最终，简化莱姆的诊断 疾病将允许较早的诊断，改善患者结局和与莱姆相关的地役 医疗保健负担。