Rapid Diagnostic Assays for Human Calciviruses

人类杯状病毒的快速诊断分析

• 批准号: 6747786
• 负责人: Robert L. Atmar
• 金额: $ 22.93万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6747786
• 关键词: Calicivirus; Norwalk virus; biosensor device; clinical research; diagnosis design /evaluation; diagnostic tests; gastroenteritis; gastrointestinal disorder diagnosis; human subject; laboratory mouse; laboratory rabbit; monoclonal antibody; polymerase chain reaction; rapid diagnosis; virus antigen

The focus of the studies proposed in this project is the development of rapid diagnostic assays for the detection of human caliciviruses. In Specific aim 1, we wilt develop broadly-reactive diagnostic reagents for human caliciviruses. Human caliciviruses are divided into two genera, Norovirus and Sapovirus, and within these genera are many genetically and antigenically distinct groups. We have previously identified monoclonal antibodies that recognize epitopes shared among the noroviruses. In addition, we and others have identified blood group antigens involved in binding to norovirus particles. We have recently confirmed the specificity of one of these antigens (Lewis d) for affinity purification of Norwalk virus. These and newly developed monoctonal antibodies and blood group carbohydrates will be characterized for use in the development of rapid, broadly-reactive diagnostic assays. In Specific aim 2, we will explore new rapid formats to detect human caliciviruses. High affinity, cross-reactive monoclonal antibodies characterized in specific aim 1 will be used in assays that require minimal sample preparation and yield a result in less than 30 minutes. Formats to be evaluated include those that have been used successfully for the detection of other human viruses and include traditional enzyme immunoassays, chromatographic immunoassays, latex agglutination assays, and dot-blot immunoassays. The assays will be tested against a panel of both human and animal caliciviruses and clinical specimens to determine sensitivity, specificity, positive and negative predictive values, and limits of detection. Developed assays will be validated by providing the diagnostic tests to other laboratories (e.g., CDC, state health labs) using RT-PCR assays to evaluate large numbers of specimens for human caliciviruses. In Specific aim 3, new approaches will be developed to correlate detection of human caliciviruses with their potential infectivity and susceptibility to inactivation. The inability to cultivate human caliciviruses in vitro has hampered assessment of their infectivity. A biosensoring system will utilize green fluorescent protein as a reporter signal that is expressed following the initiation of a viral infection in a transformed cell line. Different disinfection measures will be evaluated for both human and animal caliciviruses and the results will be correlated with those obtained by cell culture for the animal caliciviruses that are cultivatable.

该项目提出的研究重点是开发用于检测人类杯状病毒的快速诊断方法。在具体目标 1 中，我们将开发针对人类杯状病毒具有广泛反应性的诊断试剂。人类杯状病毒分为两个属：诺如病毒和沙波病毒，这些属内有许多在遗传和抗原上不同的群体。我们之前已经鉴定出可识别诺如病毒共享表位的单克隆抗体。此外，我们和其他人已经鉴定出血型抗原 参与与诺如病毒颗粒的结合。我们最近证实了其中一种抗原 (Lewis d) 对于诺瓦克病毒亲和纯化的特异性。这些和新开发的单抗体和血型碳水化合物将用于开发快速、广泛反应的诊断测定法。在具体目标 2 中，我们将探索新的快速检测人类杯状病毒的方法。以特定目标 1 为特征的高亲和力、交叉反应性单克隆抗体将用于需要最少样品制备并在 30 分钟内产生结果的测定。待评估的形式包括已成功用于检测其他人类病毒的形式，包括传统的酶免疫测定、色谱免疫测定、乳胶凝集测定和斑点印迹免疫测定。这些测定将针对一组人类和动物杯状病毒以及临床样本进行测试，以确定敏感性、特异性、阳性和阴性预测值以及检测限。开发的检测方法将通过向其他实验室（例如疾病预防控制中心、州卫生实验室）提供诊断测试来验证，使用 RT-PCR 检测来评估大 人类杯状病毒样本的数量。在具体目标 3 中，将开发新方法将人类杯状病毒的检测与其潜在感染性和灭活敏感性相关联。无法在体外培养人类杯状病毒阻碍了对其感染性的评估。生物传感系统将利用绿色荧光蛋白作为报告信号，该信号在转化细胞系中病毒感染开始后表达。将针对人类和动物杯状病毒评估不同的消毒措施 并且结果将与通过可培养的动物杯状病毒的细胞培养获得的结果相关。