Electrochemical RT activity assay for measuring HIV load

描述(由申请人提供)：在这个第一阶段的SBIR项目中，Alderon Biosciences Inc.(ABI)建议证明使用电化学逆转录酶(ERT)检测临床样本中HIV病毒载量的可行性。成功将为第二阶段的开发、FDA的批准以及使用这一先进技术的商业试剂盒的生产奠定基础。长期目标是生产一种负担得起(相当于或10美元)的艾滋病毒病毒负载测试，使用一种廉价和便携的仪器提供当天的结果。目前可用的基于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，每次检测的费用可达100-250美元。ABI的项目目标是回应对艾滋病毒病毒载量测量系统的迫切需求，该系统实用且负担得起在资源匮乏的环境中使用。ABI的低成本病毒载量分析可以提高抗逆转录病毒治疗的有效性，并在资源匮乏的环境中降低数百万感染患者的病毒传播发生率。ABI提出的新的RT检测方法的创新之处在于使用了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(但不是FDA批准的)病毒载量监测替代方法。与难以量化某些HIV亚型的基于PCR的检测方法不同，RT活性检测对HIV-1(无论是哪种亚型)和HIV-2同样有效，而且比基于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商业RT活性试剂盒中RT浓度的测量。在ABI对光学分析的适应中，RT活性将导致在传感器表面的延伸链上催化掺入许多电活性标记。一种成本极低的恒电位仪将施加电势，并通过安培法测量绑在传感器表面的电活性标签所催化的电流量。ERT分析中产生的电流将反映样品中的RT量。与北卡罗来纳大学艾滋病研究中心的合作将建立ERT检测结果与血浆艾滋病毒水平之间的相关性。第一阶段工作的可衡量的里程碑将是展示准确、定量和精确的ERT测量，与基于聚合酶链式反应的血浆艾滋病毒病毒载量和灵敏度的显著相关性(r D 0.90)低至300个病毒颗粒/毫升。

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.