An Improved Diagnostic for Lyme Arthritis

描述(由申请人提供)： 每年，数以千计的美国人感染莱姆病，这是由伯氏疏螺旋体细菌引起的。目前，莱姆病的临床诊断受到临床样本中伯氏杆菌细胞水平较低的限制。这对那些患有这种疾病的人来说是一个严重的困难，因为大多数没有得到治疗的人都会患上莱姆关节炎。莱姆关节炎是一种衰弱的疾病，表现为急性关节疼痛和肿胀的症状。在这个提案中，我们概述了一个研究项目，该项目将开发一种高度敏感的莱姆病诊断分析方法。与目前可用的其他化验方法不同，这种化验方法将直接在莱姆关节炎患者的滑液中检测伯氏杆菌细胞，从而填补目前市场上存在的一个空白。值得注意的是，数以千计的美国人患有关节炎症状，他们需要接受一系列测试，以帮助确定他们不适的根源。这项测试将为这些患者提供有价值的信息，为治疗方案提供洞察力。在这个第一阶段的提案中，我们开始开发一种灵敏的莱姆关节炎诊断方法。我们将确定高亲和力的多肽配体，通过它们与细菌外表面蛋白ErpA和ErpP的相互作用直接检测伯氏杆菌细胞。这项检测的多肽序列将来自人类补体调节因子H，它是ErpA和ErpP的已知配体。我们将使用高密度的多肽芯片选择来鉴定和亲和与ErpA和ErpP特异性结合的H因子的成熟肽序列。我们将进行伯氏杆菌全细胞结合试验，以检验为我们的诊断试验选择的每一种多肽的实用性。此外，我们将使用人类细胞进行全细胞结合分析，以证明我们的诊断探针的特异性。一旦我们确定了通过与外表面蛋白ErpA和ErpP相互作用而在全细胞结合试验中特异性检测伯氏杆菌细胞的高亲和力多肽，这些配体将进入莱姆诊断计划的第二阶段，其中将包括开发用于诊断莱姆关节炎的原型仪器。值得注意的是，我们为这一提议组建了一个强大的研究团队。首席研究员约翰·米勒博士是一位分子微生物学家，他在药物发现的多肽选择以及体内和体外生物检测的开发方面拥有丰富的经验。Sriram Shankar博士在抗体和基于多肽的分析的设计和开发方面经验丰富。布莱恩·史蒂文森博士是国际知名的伯氏杆菌微生物学家，他的研究重点是外表面蛋白ErpA和ErpP的生物学及其与人H因子的相互作用。综上所述，我们认为鉴于H因子与伯氏杆菌外表面蛋白ErpA和ErpP之间的强烈相互作用，我们将在全细胞结合实验中成功地鉴定出能够特异性检测伯氏杆菌细胞的高亲和力多肽。这些配体将作为我们新的莱姆病诊断方法中的高亲和力探针。 公共卫生相关性： 我们建议开发一种新的莱姆关节炎诊断方法，直接检测临床样本中的伯氏杆菌细胞。我们将鉴定来自人类补体调节因子H的高亲和力多肽，这些多肽将共同用作我们的诊断探针，特异性结合伯氏杆菌外表面蛋白ErpA和ErpP。这些多肽配体将为我们提供一种简便的方法来检测临床标本中的伯氏杆菌细胞。

DESCRIPTION (provided by applicant): Every year, thousands of Americans contract Lyme disease, which is caused by the bacterium Borrelia burgdorferi. Currently, clinical diagnosis of Lyme disease is limited by the low levels of B. burgdorferi cells in clinical samples. This presents a severe hardship on those who have the disease, since most individuals who go untreated develop Lyme arthritis. Lyme arthritis is a debilitating condition manifested by symptoms of acute joint pain and swelling. In this proposal, we outline a research project that will develop a highly sensitive, diagnostic assay for Lyme disease. This assay, unlike other assays that are currently available, will directly detect B. burgdorferi cells in the synovial fluid of individuals suffering from Lyme arthritis, thus filling a void that currently exists in the market. Notably, thousands of Americans who suffer with arthritic symptoms undergo a battery of tests to help pinpoint the source of their discomfort. This assay will provide valuable information to those patients, offering insight into treatment options. In this Phase I proposal, we initiate development of a sensitive diagnostic assay for Lyme arthritis. We will identify high affinity peptide ligands that directly detect B. burgdorferi cells through their interaction with the bacterial outer surface proteins ErpA and ErpP. Peptide sequences for this assay will be derived from the human complement regulator, factor H, which is a known ligand of ErpA and ErpP. We will identify and affinity mature peptide sequences of factor H that specifically bind ErpA and ErpP using high-density peptide chip selections. B. burgdorferi whole-cell binding assays will be performed to examine the utility of each of the peptides selected for our diagnostic assay. In addition, we will carry out whole-cell binding assays using human cells to demonstrate the specificity of our diagnostic probes. Once we have identified high affinity peptides that specifically detect B. burgdorferi cells in whole-cell binding assays through their interactions with outer surface proteins ErpA and ErpP, these ligands will progress to the second Phase of our Lyme Diagnostic program, which will include the development of a prototype instrument for the diagnosis of Lyme arthritis. It is worth noting that we have assembled a strong research team for this proposal. Dr. John Mueller, the Principal Investigator, is a molecular microbiologist who has extensive experience in peptide selections for drug discovery and the development of in vivo and in vitro biological assays. Dr. Sriram Shankar is experienced in the design and development of antibody and peptide-based assays. Dr Brian Stevenson is an internationally renowned B. burgdorferi microbiologist, whose research focuses on the biology of outer surface proteins ErpA and ErpP and their interaction with human factor H. In summary, we feel that given the strong interactions between factor H and the B. burgdorferi outer surface proteins ErpA and ErpP, we will successfully identify high-affinity peptides that can specifically detect B. burgdorferi cells in our whole-cell binding assay. These ligands will serve as high affinity probes in our novel diagnostic assay for Lyme disease. PUBLIC HEALTH RELEVANCE: We propose to develop a novel diagnostic assay for Lyme arthritis that will directly detect B. burgdorferi cells in clinical samples. We will identify high affinity peptides derived from human complement regulator, factor H, that will collectively serve as our diagnostic probe to specifically bind B. burgdorferi outer surface proteins ErpA and ErpP. These peptide ligands will afford us a facile assay to detect B. burgdorferi cells in clinical samples.