Hybrid ELISA: Simple and specific one-tier assay for Lyme disease

摘要 莱姆病，由感染螺旋体伯氏疏螺旋体或密切相关的物种，是最 在美国常见的病媒传播疾病，据统计， 最近的公共卫生评估。它也是为数不多的需要实验室两步走的传染病之一 测试方案，包括筛选测定，通常是ELISA，然后是确认性免疫印迹。这 当疾病进展超过标志性游走性红斑皮疹或皮疹时， 不能确定，症状或病史提示莱姆病。两步协议是一个结果， 大多数莱姆病筛查试验的特异性差，导致假阳性率高。这反过来又是由于 许多细菌抗原的交叉反应性质，结合常规ELISA的局限性 免疫化学原则上，在开始治疗之前必须获得确认结果，但在实践中， 在这一过程中经常发生的多日延迟经常导致医生开抗生素处方 在没有明确的实验室结果的情况下进行治疗。作为每年超过三百万的莱姆中的绝大多数 在美国，测试是在最终发现没有莱姆病的人身上进行的，这种做法 导致不必要的处方和使用抗生素，导致抗生素耐药性的增长 这已成为对公共卫生的重大威胁。具有显著更高特异性的第一步测试将 避免了第二步测试的需要，并使临床医生能够及时做出明智的治疗决定。 以可靠的测试结果为基础。 该项目旨在通过减少莱姆病检测， 将目前的两步法改为一步法。为了实现这一目标，我们开发了一种新的ELISA 莱姆病抗体检测的方法，使异常高的测定特异性。测试是基于 在一个独特的免疫化学形式的良好建立，高度特异性和敏感的疏螺旋体抗原。的 新型ELISA免疫化学消除了几乎所有的非特异性反应， 特异性，但灵敏度高于传统的两级测试方案。 因此，该检测方法有望为莱姆病提供一步式检测解决方案， 在科学和监管层面，替代最初两步法的方法正获得合法性。 在第一阶段，我们开发了一种原型ELISA测定法，通过展示更高的灵敏度和更高的灵敏度来证明其可行性。 与两种方法相比，对包括莱姆病患者和对照组的回顾性样本的特异性相当， 层测试结果。在第二阶段，我们将完成检测试剂盒的商业化开发， 前瞻性和回顾性临床研究，并向FDA提交510（k）申请，以使用ELISA作为 莱姆病的一步测试，批准后即可进行商业投放。

Abstract Lyme disease, caused by infection with the spirochete Borrelia burgdorferi or closely related species, is the most common vector-borne disease in the United States, accounting for nearly 500,000 infections per year according to recent public health estimates. It is also one of the few infectious diseases that requires a two-step laboratory testing protocol, comprising a screening assay, typically ELISA, followed by a confirmatory immunoblot. This protocol is followed where the disease has progressed beyond the hallmark erythema migrans rash, or the rash cannot be identified, and symptoms or history suggest Lyme disease. The two-step protocol is a result of the poor specificity of most Lyme screening tests, which lead to a high false positive rate. This in turn is due to the cross-reactive nature of many bacterial antigens, combined with the limitations of conventional ELISA immunochemistry. In principle, a confirmatory result must be obtained prior to initiating treatment, but in practice, the multi-day delay that is often incurred in this process frequently leads physicians to prescribe antibiotic treatment in the absence of definitive lab results. As the vast majority of the more than three million annual Lyme tests in the U.S. are carried out on individuals who are ultimately found not to have Lyme disease, this practice leads to the unnecessary prescription and use of antibiotics, contributing to the growth of antibiotic resistance which has become a significant threat to public health. A first step test with significantly higher specificity would avoid the need for a second step test and enable clinicians to make informed treatment decisions in a timely manner, based on credible test results. This project is aimed at bringing about a significant change in medical practice by reducing Lyme testing from the current two-step process to a one-step process. To achieve this objective, we have developed a novel ELISA methodology for Lyme antibody detection that enables exceptionally high assay specificity. The test is based on well-established, highly specific and sensitive Borrelia antigens in a unique immunochemical format. The novel ELISA immunochemistry eliminates almost all non-specific reactivity, yielding results comparable in specificity but higher in sensitivity than those obtained with the conventional two-tier testing protocol. Consequently, this assay promises to deliver a one-step testing solution for Lyme disease, at a time when alternatives to the original two-step method are gaining legitimacy at the scientific as well as regulatory level. In Phase I, we developed a prototype ELISA assay, proving feasibility by demonstrating higher sensitivity and equivalent specificity on retrospective samples comprising Lyme patients and controls, in comparison with two- tier testing results. In Phase II, we will complete development of the assay into a commercial product, carry out prospective and retrospective clinical studies and submit a 510(k) application to FDA for use of the ELISA as a one-step test for Lyme disease, enabling commercial launch upon approval.