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: 8876556
• 负责人: Michel Ledizet
• 金额: $ 82.57万
• 依托单位: L2 DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8876556
• 关键词: Antibiotics; Antibodies; Antibody Response; 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; Health; 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; 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; accurate diagnosis; base; cross reactivity; diagnostic assay; disease diagnosis; erythema migrans; improved; in vivo; pathogen; phase 1 study; protein expression; 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）：当前对莱姆病的测试是次优的，因为它需要两个测定平台，其中包括一个对结果的主观解释，并且因为它在疾病的早期阶段具有较低的敏感性。莱姆病的病因伯氏伯氏菌感染是通过ixodid tick的咬伤传播的，并导致只有短暂性血液生物阶段的病原体负担均匀，因此在易于获得的血液中，细菌直接检测的直接检测敏感性（如血液）很低。因此，诊断测试依赖于血清学测定来检测细菌的抗体。 CDC当前建议使用初始ELISA进行两层测定，并且必须通过免疫印迹确认阳性或模棱两可的结果。从历史上看，这两种测定法都是用整个细胞进行体外培养细菌进行的，尽管现在众所周知，B. burgdorferi螺旋体从移动到哺乳动物宿主的滴答和体外培养时会改变其蛋白质表达。最近，已经开发了来自VLSE蛋白的C6肽的ELISA，现在有时被使用而不是裂解物ELISA。该测定的结果仅基于一种抗原，但仍经常被耗时，主观和昂贵的免疫印迹所证实。我们使用Luminex XMAP技术平台开发了一种新的一步快速多路复用测定，该测定平台在当前测试的测定平台和所使用的抗原上都改进了。我们确定了哺乳动物宿主中螺旋体表达的一组抗原，并产生了早期IgG反应。我们已经开发了一种比ELISA或免疫印迹更大的动态范围的多重测定法，并且可以更快，客观地完成。多种但特定的抗原的使用也消除了对两层测试的需求。我们第一阶段研究的初步结果还表明，我们的测定方法提高了检测早期感染的敏感性，尤其是在当前测定失败的情况下。