Ultrasensitive Detection of Hepatitis Viruses by Immunoassay Amplification

描述（由申请人提供）：该提案响应了美国国立卫生研究院最近发起的抗击病毒性肝炎沉默流行病的行动计划，重点关注肝炎的早期诊断，这对于预防进一步感染和改善治疗至关重要。大约有1.7亿人感染HCV，其中许多人发展为肝硬化和肝癌。很大一部分慢性感染者没有表现出症状，因此不知道他们患癌症的风险，这突出表明需要进行全面监测。目前，HCV感染早期的诊断是使用核酸扩增检测（NAT）-金标准。然而，NAT不适合于调查大的人群或用于现场护理（POC）测试，因为它是昂贵的并且需要昂贵的设备和训练有素的人员。BCR诊断公司(BCR)提出了一种具有成本效益的替代NAT的应用其创新的生物传感器技术，以降低低检测限（LDL）的珠为基础的酶联免疫吸附测定（ELISA）的HCV核心蛋白（cAg）。BCR技术利用表型工程化的荧光细菌孢子（F-spores（tm））作为纳米检测器。所提出的生物传感器基于现有的生物芯片（称为80 K-bioChip（TM）），其是包含80，000个独立的生物传感器（称为微滤器（r））的一次性装置，每个生物传感器填充有约200个F-孢子。80 K-bioChip的一个特点是使用廉价的数码相机作为定量读数。该项目有以下具体目标：具体目标1：验证用于测量人血清中低水平HCV核心蛋白（cAg）的BCR生物传感器。方法：将使用加标不同浓度cAg的人血清检测生物传感器性能。里程碑：数据显示生物传感器显著改善了cAg常规ELISA的LDL。特异性AIM 2：使用HCV血清转化板测量生物传感器的灵敏度和特异性。方法：该生物传感器将用于测量来自HCV感染的各个不同阶段的患者的商业血清转换面板中的个体血清的cAg水平。里程碑：数据显示灵敏度和特异性接近100%。第二阶段计划：将生物传感器转换为可用于研究和临床实验室的可销售原型，并将原型用于旨在获得美国食品药品监督管理局（FDA）510（k）许可的研究。项目描述

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