Novel Point-of-Care Multiplex Lyme Disease Test

描述（由申请人提供）：莱姆病（LD）是由螺旋体属的螺旋体细菌引起的。伯氏疏螺旋体是美国莱姆病的主要病因，而阿夫泽利疏螺旋体和加里氏疏螺旋体在大多数欧洲病例中都有牵连。美国疾病控制与预防中心仅在2007年就报告了27,444例莱姆病病例。据估计，每年进行250万次LD血清学检测。为了响应NIAID在开发莱姆病诊断新方法方面的请求，并解决当前LD检测的局限性，我们建议开发一种高灵敏度的多点手持诊断设备，能够检测人类血清、唾液和尿液中针对伯氏疏螺旋体的IgM和IgG抗体的存在。我们设备的技术基于免疫沉淀原理，类似于流行的妊娠试验。横向流动色谱和流动通过测试提供快速检测的关键成分，用于POC测试。该方法的关键特点是能够将抗体或抗原共价地附着在高度着色的纳米级胶体金上，从而达到高灵敏度。我们的横向流动测试分析开发经验表明，该分析与ELISA一样敏感，同时将分析时间从8小时缩短到5分钟。拟议测试的另一个关键特征是收集14种体内诱导的伯氏疏螺旋体特异性非变性抗原，这些抗原已被证明在人体中引起强烈的抗体反应。这些抗原将用于捕获人血清样本中出现的人伯氏疏螺旋体抗体。在第一阶段，我们将使用伯氏疏螺旋体的重组抗原，在第二阶段，我们将使用所有三种伯氏疏螺旋体——伯氏疏螺旋体、阿夫泽利伯氏疏螺旋体和加里尼伯氏疏螺旋体的重组蛋白来进一步提高灵敏度。POC分析将是廉价的，每次测试不到20美元，稳健，只需要一个步骤，现场能力，用户友好，温度稳定，不需要样品处理。结果可以在10到30分钟内读取。公共卫生相关性：莱姆病（LD）是最广泛的蜱传疾病，仅2007年在美国就影响了27,000多人。目前的LD诊断测试既耗时又昂贵。我们建议开发一种类似于普通妊娠试验的高精度即时检测方法。它价格低廉，每次测试不到20美元，功能强大，只需要一个步骤，用户友好，温度稳定，不需要样品处理。结果可以在10到30分钟内读取。

DESCRIPTION (provided by applicant): Lyme disease (LD) is caused by spirochetal bacteria from the genus Borrelia. Borrelia burgdorferi is the predominant cause of Lyme disease in the United States, whereas Borrelia afzelii and Borrelia garinii are implicated in most European cases. CDC reported 27,444 cases of Lyme disease in the United State in 2007 alone. It is estimated 2.5 million LD serological tests have been done annually. In response to the solicitation from NIAID in developing novel approaches for the diagnosis of Lyme disease, and to address the limitation of current LD assays, we propose to develop a highly sensitive Point-of-Care Multiplex Hand-Held Diagnostic Device capable of detecting the presence IgM and IgG antibodies against Borrelia species proteins in human serum, human saliva and human urine. The technology of our device is based on immunoprecipitation principles similar to those found in popular pregnancy tests. Lateral-flow chromatographic and flow-through tests offer fast detection of critical components for use in POC testing. The key feature of a proposed flow- through tests to achieve high sensitivity is the ability to covalently attach antibodies or antigens to highly intensely colored, nanometer-sized colloidal gold. Our lateral flow test assay development experience has demonstrated that the assay is as sensitive as ELISA, while reducing assay time from 8hrs to 5 minutes. Another key feature of the proposed test is the collection of 14 Borrelia specific in vivo-induced, non-denatured antigens that have been shown to elicit strong antibody response in humans. These antigens will be used to capture human Borrelia antibodies presented in human serum samples. In Phase I, we will use recombinant antigens of B. burgdorferi species, in Phase II, for each antigen we will use recombinant proteins from all three Borrelia species - B. burgdoferi, B. afzelii and B. garinii to further increase sensitivity. The POC assay will be inexpensive, less than $20 per test, robust, requires only a single step, field-capable, user-friendly, and temperature stable, with no requirement for sample processing. The results can be read in 10 to 30 minutes. PUBLIC HEALTH RELEVANCE: Lyme disease (LD) is the most widespread tick-borne disease affecting more than 27,000 people in the US in 2007 alone. The current LD diagnosis tests are both time consuming and expensive. We propose to develop a high accuracy Point-of-Care test similar to common pregnancy test. It is inexpensive, less than $20 per test, robust, requires only a single step, user-friendly, and temperature stable, with no requirement for sample processing. The results can be read in 10 to 30 minutes.