Diagnostic Assays for early Lyme Borreliosis using in-vivo expressed antigens

使用体内表达抗原诊断早期莱姆疏螺旋体病

• 批准号: 8782201
• 负责人: Michel Ledizet
• 金额: $ 82.94万
• 依托单位: L2 DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8782201
• 关键词: Antibiotics; Antibodies; Antibody Formation; Antigens; Bacteria; Bacterial Antigens; Binding; Biological Assay; Bite; Black-legged Tick; Blood; Body Fluids; Borrelia burgdorferi; Case Study; Cells; Centers for Disease Control and Prevention (U.S.); Clinical; Detection; Diagnosis; Diagnostic; Diagnostic tests; Disease; Early Diagnosis; Enzyme-Linked Immunosorbent Assay; Epitopes; Immunoblotting; Immunoglobulin G; Immunoglobulin M; In Vitro; Infection; Lyme Disease; Measurable; Medical; Methods; Molecular Weight; Movement; One-Step dentin bonding system; Order Spirochaetales; Patients; Pattern; Peptides; Performance; Phase; Physicians; Plague; Proteins; Public Health; Reading; Reliance; Reporting; Sampling; Sensitivity and Specificity; Serologic tests; Serological; Serum; Skin; Specificity; Staging; Step Tests; Symptoms; Syndrome; Technology; Testing; Ticks; Time; Tissues; United States; Vector-transmitted infectious disease; Work; base; cross reactivity; disease diagnosis; erythema migrans; improved; in vivo; pathogen; phase 1 study; protein expression; public health relevance; research study; response

DESCRIPTION (provided by applicant): Current testing for Lyme disease is suboptimal because it requires two assay platforms, including one with subjective interpretation of results, and because it has lower sensitivity in the early stage of the illness. Infection with Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the bite of an Ixodid tick and results in uniformly low pathogen burdens with only a transient bloodborne phase, so that the sensitivity of direct detection assays of the bacteria in easily accessible tissues such as the blood is low. Diagnostic testing therefore relies on serologic assays to detect antibodies to the bacteria. The CDC currently recommends a two-tier assay with an initial ELISA, and positive or equivocal results must be confirmed by immunoblot. Historically both assays were performed with whole cell sonicate of in vitro cultured bacteria even though it is now known that B. burgdorferi spirochetes alter their protein expression when they move from the tick to the mammalian host and when they are cultured in vitro. More recently an ELISA for the C6 peptide from the VlsE protein has been developed and is now sometimes used instead of the lysate ELISA. The results of this assay, which is based on just one antigen, are still often confirmed by immunoblot that is time consuming, subjective, and expensive. We have developed a new one-step rapid multiplex assay using the Luminex xMAP Technology platform that improves upon both the assay platform of the current tests and the antigens used. We identified a panel of antigens that are expressed by spirochetes within the mammalian host and to which early IgG responses are generated. We have developed a multiplex assay that has a much larger dynamic range than ELISA or immunoblot, and can be completed more rapidly and objectively. The use of multiple but specific antigens also obviates the need for two-tier testing. Preliminary results from our Phase I study also suggest that our assay has improved sensitivity for detecting early infections, particularly when the current assays fail.

描述（由申请人提供）：目前对莱姆病的检测是次优的，因为它需要两个检测平台，其中一个对结果有主观解释，并且在疾病的早期阶段灵敏度较低。莱姆病的病原体伯氏疏螺旋体的感染是通过伊蚊蜱的叮咬传播的，导致病原体负荷一致较低，只有短暂的血源性阶段，因此，对容易接触的组织（如血液）中的细菌进行直接检测的灵敏度较低。因此，诊断测试依靠血清学分析来检测细菌抗体。美国疾病控制与预防中心（CDC）目前建议采用两级检测，初始ELISA，阳性或模棱两可的结果必须通过免疫印迹证实。历史上，这两项检测都是用体外培养细菌的全细胞超声进行的，尽管现在已经知道，当伯氏螺旋体从蜱虫转移到哺乳动物宿主以及在体外培养时，它们的蛋白质表达会发生改变。最近，从VlsE蛋白中提取C6肽的酶联免疫吸附试验已经被开发出来，现在有时用来代替酶联免疫吸附试验。这种检测结果仅基于一种抗原，通常仍需通过耗时、主观且昂贵的免疫印迹来证实。我们使用Luminex xMAP技术平台开发了一种新的一步快速多重检测，改进了当前测试的检测平台和使用的抗原。我们确定了一组抗原，由哺乳动物宿主内的螺旋体表达，并对其产生早期IgG反应。我们开发了一种多重检测方法，其动态范围比ELISA或免疫印迹大得多，并且可以更快、更客观地完成。使用多种但具有特异性的抗原也避免了两层检测的需要。我们一期研究的初步结果还表明，我们的检测方法提高了检测早期感染的灵敏度，特别是在当前检测方法失败的情况下。