Electrochemical RT activity assay for measuring HIV load

用于测量 HIV 负荷的电化学 RT 活性测定

• 批准号: 7031579
• 负责人: ROBERT W HENKENS
• 金额: $ 30万
• 依托单位: ALDERON BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031579
• 关键词: AIDS; AIDS /HIV test; HIV infections; RNA directed DNA polymerase; bioassay; biomedical equipment development; cost effectiveness; diagnosis design /evaluation; electrochemistry; enzyme activity; human immunodeficiency virus 1; polymerase chain reaction; portable biomedical equipment; rapid diagnosis; virus genetics; virus load

DESCRIPTION (provided by applicant): In this Phase I SBIR project Alderon Biosciences Inc. (ABI) proposes to demonstrate the feasibility of using electrochemical reverse transcriptase (eRT) assays for determinations of HIV viral load in clinical samples. Success will set the stage for Phase II development, FDA approval, and production of commercial kits using this advanced technology. The long-range goal is to produce an affordable (equal to or <$10) HIV viral load test that gives same-day results using an inexpensive and portable instrument. Currently available FDAapproved PCR-based tests for determination of HIV viral load require expensive instrumentation, utilize complex molecular biology procedures, and can cost $100-$250 per test. ABI's project goal is a response to the urgent need for a HIV viral load measurement system that is practical and affordable for use in resource-poor settings. ABI's lower-cost viral load assays could increase the effectiveness of antiretroviral therapy and decrease the incidence of virus transmission for millions of infected patients in resource-poor settings. The innovation in the new RT assays proposed by ABI lies in the use of advanced electrochemical detection methods for quantifying RT activity. RT assays are a recognized (but not FDA-approved) alternative method for viral load monitoring. Unlike PCR-based assays that have problems quantifying some HIV subtypes, RT activity tests work equally well for HIV-1 (regardless of subtype) and HIV-2 and are less subject to problems of contamination than PCR-based assays. Formation of an optically detectable product is a measure of RT concentration in commercially available RT-activity kits. In ABI's adaptation of the optical assays, RT activity will result in catalytic incorporation of many electroactive labels on extended chains at a sensor surface. An extremely low-cost potentiostat will apply a potential and amperometrically measure the amount of current catalyzed by the electroactive labels tethered to the sensor surface. The current generated in the eRT assays will reflect the amount of RT in the sample. Collaborative work with the UNC Center for AIDS Research will establish the correlation of the eRT assay results with plasma levels of HIV. The measurable milestone of Phase I work will be demonstration of accurate, quantitative and precise eRT measurements with significant correlation (r D 0.90) to PCR-based determinations of plasma HIV viral load and sensitivity down to 300 virus particles/mL.

描述（由申请人提供）： 在第一阶段SBIR项目中，Alderon Biosciences Inc. (ABI)提出证明使用电化学逆转录酶（eRT）测定法测定临床样本中HIV病毒载量的可行性。成功将为II期开发、FDA批准和使用这种先进技术生产商业试剂盒奠定基础。长期目标是生产一种负担得起的（等于或<10美元）艾滋病毒载量测试，使用廉价和便携式仪器提供当天的结果。目前FDA批准的用于确定HIV病毒载量的基于PCR的测试需要昂贵的仪器，利用复杂的分子生物学程序，并且每次测试的成本为100 - 250美元。ABI的项目目标是对迫切需要一个艾滋病毒载量测量系统的反应，该系统实用且可负担得起，可用于资源匮乏的环境。ABI的低成本病毒载量检测可以提高抗逆转录病毒治疗的有效性，并减少资源匮乏环境中数百万感染患者的病毒传播发生率。ABI提出的新RT检测方法的创新在于使用先进的电化学检测方法来定量RT活性。RT检测是一种公认的（但未经FDA批准的）病毒载量监测替代方法。与基于PCR的检测方法存在量化某些HIV亚型的问题不同，RT活性检测方法对HIV-1（无论亚型如何）和HIV-2同样有效，并且比基于PCR的检测方法更少受到污染问题的影响。光学可检测产物的形成是市售RT活性试剂盒中RT浓度的量度。在ABI的适应的光学测定，RT活动将导致在传感器表面的延伸链上的许多电活性标记的催化掺入。一个极低成本的恒电位仪将施加一个电势，并通过安培计测量由拴在传感器表面的电活性标签催化的电流量。eRT测定中产生的电流将反映样品中的RT量。与美国艾滋病研究中心的合作将建立eRT检测结果与血浆HIV水平的相关性。I期工作的可测量里程碑将是证明准确、定量和精确的eRT测量与基于PCR的血浆HIV病毒载量测定具有显著相关性（r D 0.90），灵敏度低至300个病毒颗粒/mL。