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

摘要 莱姆疏螺旋体病是美国主要的媒介传播疾病。早期诊断和治疗 莱姆病对预防严重并发症的发展至关重要，如广泛性关节炎、慢性 神经损伤和心脏炎。临床上的一个关键障碍是无法诊断莱姆病 感染早期未出现明显红斑移行的患者。唯一一个FDA批准的测试 对于莱姆病是两级血清学检测，执行起来很费力。更重要的是，这种化验 由于患者发展所需的时间，无法在早期阶段诊断莱姆病 对感染莱姆病病原体伯氏疏螺旋体(BB)的抗体反应。在这 我们的目标是鉴定和验证样本中的尿液和血清特异性抗原。 从莱姆病患者那里收集的。作为未来提案的一部分，经过验证的抗原将用于 建立用于莱姆病早期诊断的BB抗原检测方法。这是一项具有挑战性的研究 我们认为，这一努力需要坚定不移地关注以下两个假设：第一，发展 仅检测一种BB抗原的诊断试验不会导致对早期莱姆病的临床有用的试验 疾病。我们强烈认为，将需要以多种抗原为靶点，而不是单一抗原。 用于开发一种高度灵敏的分析方法。在我们的初步研究中，我们优化了方法以 发现莱姆病患者样本中存在的抗原以及壁虱传播的 莱姆病的猕猴模型。在血清中发现了六种候选的BB抗原；这些抗原将 通过这份R21提案考虑在患者样本中进行验证。以演示如何使用 本提案中描述的DIA-MS方法将导致对潜在抗原结果的一致检测 在一项初步研究中，使用所提出的方法对5个急性患者血清样本进行了分析。 分析发现，在所有5个样本中都检测到243种蛋白质，证实了该方法的可靠性。 从生物样本中可靠地检测到BB特异性抗原是一项重大的诊断突破， 然而，多种莱姆抗原的检测将是获得高临床敏感性的关键。第二，一个 对早期莱姆病的诊断测试将需要在下游化验之前对患者样本进行浓缩。这 从我们对莱姆样本的初步研究以及我们对其他 病人样本中的感染性病原体。样品浓缩协议的开发需要最低限度的 处理将确保浓缩与临床实验室的工作流程兼容。一次合作 约翰霍普金斯莱姆病研究中心将允许包括配对的人类尿液和血清 项目发现和验证阶段的样本。最终，简化莱姆病的诊断 疾病将有助于更早的诊断，改善患者的预后，并缓解与莱姆相关的疾病 医疗保健负担。

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.