Ultrasensitive Detection of Hepatitis Viruses by Immunoassay Amplification

说明(申请人提供)：本提案响应国家卫生研究院最近发起的抗击病毒性肝炎静止性流行的行动计划，重点放在肝炎的早期诊断上，这对防止进一步感染和改进治疗至关重要。大约有1.7亿人感染了丙型肝炎病毒，其中许多人发展为肝硬变和肝癌。很大一部分慢性感染者没有表现出症状，因此没有意识到他们患癌症的风险，这突显了全面监测的必要性。目前，丙型肝炎病毒感染早期阶段的诊断是使用核酸扩增检测(NAT)--这是金标准。但是，NAT不适合调查大量人口或进行护理点(POC)测试，因为它价格昂贵，需要昂贵的设备和训练有素的人员。BCR诊断公司(BCR)通过应用其创新的生物传感器技术来降低基于微珠的酶联免疫吸附分析(ELISA)对丙型肝炎病毒核心蛋白(CAG)的低检测下限，从而提出了一种替代NAT的具有成本效益的替代方案。BCR技术利用表型工程产生的荧光细菌孢子(F-孢子(Tm))作为纳米探测器。提出的生物传感器是基于现有的生物芯片(称为80K-BioChip(Tm))，这是一种一次性设备，包含80,000个独立的生物传感器(称为微柱式生物传感器(R))，每个传感器填充约200个F-孢子。80K-生物芯片的一个特点是使用廉价的数码相机作为定量读数。具体目标1：验证BCR生物传感器用于检测人血清中低水平的丙型肝炎病毒核心蛋白(CAg)。方法：使用添加不同浓度CAG的人血清来测试生物传感器的性能。里程碑：数据显示，该生物传感器显著改善了CAG常规ELISA的低密度脂蛋白。特定目的2：使用丙型肝炎病毒血清转换板测量生物传感器的灵敏度和特异性。方法：该生物传感器将被用来测量商业血清转换板中来自不同丙型肝炎病毒感染阶段患者的单个血清的CAg水平。里程碑：数据显示敏感性和特异性接近100%。第二阶段计划：将生物传感器转化为研究和临床实验室的可销售原型，并将该原型用于一项旨在获得食品和药物管理局(FDA)510(K)批准的研究。项目说明

DESCRIPTION (provided by applicant): This proposal responds to the action plan for combating the Silent Epidemic of Viral Hepatitis recently launched by the National Institutes of Health by focusing on early diagnosis of hepatitis, which is crucial for preventing further infections and to improve therapy. Approximately 170 million people are infected with HCV, many of whom develop cirrhosis and liver cancer. A large proportion of chronically infected individuals do not exhibit symptoms, thus are unaware of their risk of developing cancer, highlighting the need for comprehensive surveillance. Diagnosis during the early stages of HCV infection is currently done using nucleic acid amplification testing (NAT)-the gold standard. However, NAT is not suitable for surveying large populations or for point of- care (POC) testing because it is expensive and requires costly equipment and trained personnel. BCR Diagnostics, Inc. (BCR) proposes a cost-effective alternative to NAT by applying its innovative biosensor technology to reduce the low detection limit (LDL) of bead-based enzyme-linked immunosorbent assays (ELISAs) for HCV core protein (cAg). BCR technology exploits phenotypically engineered fluorogenic bacterial spores (F-spores(tm)) as nanodetectors. The proposed biosensor is based on an existing biochip (termed 80K-bioChip(tm)), which is a disposable device containing 80,000 independent biosensors (termed micro-colanders(r)) that are each filled with about 200 F-spores. A feature of the 80K-bioChip is the use of an inexpensive digital camera as a quantitative readout. The project has the following specific aims: Specific AIM 1: To validate the BCR biosensor for measuring low levels of HCV core protein (cAg) in human serum. Approach: The biosensor performance will be tested using human serum spiked with different concentrations of cAg. Milestone: Data showing that the biosensor significantly improves the LDL of a conventional ELISA for cAg. Specific AIM 2: Measure sensitivity and specificity of the biosensor using HCV seroconversion panels. Approach: The biosensor will be used to measure cAg levels of individual sera in a commercial seroconversion panel from patients at various different stages of HCV infection. Milestone: Data showing sensitivity and specificity near 100%. Phase II plan: To convert the biosensor into a marketable prototype for research and clinical laboratories, and to use the prototype for a study designed to obtain 510 (k) clearance from the Food and Drug Administration (FDA). Project Description