Electrochemical RT activity assay for measuring HIV load

描述（由申请人提供）：在这个I期SBIR项目中，Alderon Biosciences Inc. （ABI）提议证明使用电化学逆转录酶（eRT）测定临床样品中HIV病毒载量的可行性。成功将为二期开发、FDA批准和使用这种先进技术的商业工具包的生产奠定基础。长期目标是生产一种负担得起的（等于或小于10美元）艾滋病毒载量检测方法，使用廉价和便携式仪器可在当天得出结果。目前，fda批准的基于pcr的HIV病毒载量检测需要昂贵的仪器，使用复杂的分子生物学程序，每次检测的费用为100- 250美元。ABI的项目目标是响应对HIV病毒载量测量系统的迫切需求，该系统实用且价格合理，可用于资源贫乏的环境。ABI的低成本病毒载量测定可以提高抗逆转录病毒治疗的有效性，并降低资源贫乏环境中数百万受感染患者的病毒传播发生率。ABI提出的新RT检测方法的创新之处在于使用先进的电化学检测方法来定量RT活性。RT测定法是公认的（但未经fda批准的）病毒载量监测的替代方法。与基于聚合酶链反应的分析方法不同，RT活性测试对HIV-1（无论何种亚型）和HIV-2都同样有效，并且比基于聚合酶链反应的分析方法更少受到污染问题的影响。在市售的RT活性试剂盒中，光学可检测产物的形成是RT浓度的量度。在ABI对光学分析的适应中，RT活性将导致许多电活性标签在传感器表面延伸链上的催化结合。一个极低成本的恒电位器将施加一个电位，并以电流计测量拴在传感器表面的电活性标签催化的电流量。在eRT测定中产生的电流将反映样品中RT的量。与北卡罗来纳大学艾滋病研究中心的合作将建立eRT测定结果与血浆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病毒颗粒/mL。

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.