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

摘要 莱姆病是由伯氏疏螺旋体或其近缘物种感染引起的 美国最常见的媒介传播疾病，每年造成30多万人感染 根据公共卫生估计。它也是为数不多的需要两步操作的传染病之一。 实验室检测方案，包括筛查分析，通常是酶联免疫吸附试验，然后是确认性检测 免疫印迹。如果疾病的进展超出了标志性红斑，则遵循该方案。 迁徙者皮疹，或皮疹无法识别，症状或病史提示莱姆病。两步走 协议是大多数莱姆筛查试验特异性差的结果，这导致了高假阳性 费率。这反过来又是由于许多细菌抗原的交叉反应性质，再加上局限性 常规的酶联免疫化学。原则上，在启动之前必须获得确认性结果。 治疗，但在实践中，往往为此目的而导致的数天延误导致医生 在没有明确的实验室结果的情况下经常开抗生素治疗。因为绝大多数人 在美国，每年大约有300万例莱姆检测是在没有莱姆的个人身上进行的 这种做法会导致不必要的处方和抗生素的使用，从而导致 抗生素耐药性已成为对公众健康的重大威胁。重要的第一步测试 更高的特异性将避免第二步测试的需要，并使临床医生能够进行知情的治疗 根据可信的测试结果，及时做出决定。 该项目旨在通过减少莱姆测试来显著改变医疗实践 从目前的两步过程转变为一步过程。为了实现这一目标，我们开发了一种新的 莱姆抗体检测的ELISA法，可实现异常高的检测特异性。测试是 基于独特免疫化学形式的成熟、高度特异和敏感的疏螺旋体抗原。 新的ELISA免疫化学法消除了几乎所有的非特异性反应，产生了与 灵敏度和特异度均高于传统的两层检测方案。因此， 这一检测方法承诺为莱姆病提供一步检测解决方案，而此时 最初的两步法在科学和监管层面都获得了合法性。 在这个第一阶段的项目中，我们将开发一个原型的酶联免疫吸附试验，通过证明敏感性来证明其可行性。 对包括莱姆病患者和对照的回溯性样本的特异性，与两级比较 检测结果。在第二阶段，我们将完成商业产品的开发，进行预期和 回顾临床研究，并向FDA提交体外诊断声明申请，允许 批准后即可商业发射。

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 over 300,000 infections per year according to 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 results in 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 for this purpose leads to physicians frequently prescribing antibiotic treatment in the absence of definitive lab results. As the vast majority of the roughly three million annual Lyme tests in the U.S. are carried out on individuals who do not 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 both sensitivity and specificity to 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 this Phase I project, we will develop a prototype ELISA assay, proving feasibility by demonstrating sensitivity and specificity on retrospective samples comprising Lyme patients and controls, in comparison with two-tier testing results. In Phase II, we will complete development of a commercial product, carry out prospective and retrospective clinical studies and submit an application to FDA for in vitro diagnostic claims, allowing commercial launch upon approval.