A lateral flow CD4 counting assay for resource-poor regions

描述（由申请人提供）：目前对hiv感染者CD4+细胞计数常规测量的需求是巨大的。目前，CD4+计数的费用是一个主要问题（特别是）在资源贫乏的环境中，那里的感染率最高。在资源贫乏的环境中，获得准确CD4+计数的成本并不是常规CD4+监测的唯一障碍。需要培训人员使用相对复杂的设备（细胞仪、细胞分选器、平板阅读器），这些设备需要一个受控的环境（在高湿和非标准温度环境中维护机器是有问题的）。该提案旨在开发一种快速CD4+计数检测方法，以解决这些问题。该检测将结合当前的护理点（POC）方法（“横向流动”）和专有且廉价的制造抗cd4 +“贪婪”捕获试剂的方法。该分析将能够在资源贫乏的环境中进行，不需要昂贵的设备，相对于环境变量是稳健的，并且可以由非熟练人员执行和解释。我们建议可以制造一种具有这些特性的准确的CD4+计数检测方法，最终用户每次检测的成本低于1美元。该分析成本估算部分基于两个假设：1)用于该分析的所有试剂将在内部开发，以规避定价中内置的试剂许可费；2)使用公共领域程序组合进行分析构建（例如试剂的喷墨沉积和可用的硝化纤维素分析膜），以避免技术许可的必要性。从技术角度来看，我们需要证明我们的“贪婪”抗CD4+捕获试剂显示CD4+表达T细胞的解离常数接近零，因此能够捕获CD4+细胞。全世界艾滋病毒阳性患者总数超过4000万。绝大多数患有这种疾病的人生活在资源贫乏的环境中，由于缺乏训练有素的人员，传统的CD4计数既昂贵又具有技术挑战性。目前正在进行的治疗工作，如世界卫生组织的“三五计划”，将在今后几年向世界上这些地区的数百万患者提供高效抗逆转录病毒疗法（如高效抗逆转录病毒疗法）。正是在这种资源贫乏的环境中，CD4计数可以说是最重要的。目前的成本和分析的复杂性限制了这一点。准确的CD4计数可用于：促进艾滋病监测；监测艾滋病的进展速度，确定何时需要治疗以防止机会性感染，在治疗前将未接触药物的患者分组，并监测抗逆转录病毒治疗的效果。目前建议每3-6个月对每个艾滋病毒感染者进行CD4检测，并根据情况增加频率。从解决技术困难和成本要求的角度来看，这里描述的分析旨在满足这一需求。目前认为，缺乏这种检测方法会导致机会性感染导致不必要的死亡。根据这一第一阶段建议正在开发的快速化验方法如果成功，将对发展中国家为接受抗逆转录病毒治疗的艾滋病毒感染者检测机会性感染（从而进行适当治疗）的能力产生巨大影响。

DESCRIPTION (provided by applicant): The current need for routine measurement CD4+ cell counts of HIV-infected individual's is immense. Currently, the cost of CD4+ counting is a major issue (especially) in resource-poor environments, where the infection rates are highest. Cost of obtaining an accurate CD4+ count is not the only barrier to routine CD4+ monitoring in resource poor settings. Personnel need to be trained to use relatively complex equipment (cytometers, cell sorters, plate readers) and such equipment needs a controlled environment (machine maintenance in high humidity and non-standard temperature environments is problematic). This proposal is aimed at development of a rapid CD4+ count assay that addresses these concerns. The assay will combine current point-of-care (POC) methodologies ("lateral flow") with a proprietary and inexpensive means of creating anti-CD4+ "avidity" capture reagents. This assay will be capable of being performed in resource-poor environments, requires no expensive equipment, is robust with respect to environmental variables, and can be executed and interpreted by unskilled personnel. We suggest that an accurate CD4+ counting assay with these attributes could be manufactured that would cost the end-user less than US $1.00 per test. This assay cost estimate is partially based on two assumptions: 1) all reagents for the assay will be developed in-house to circumvent reagent license fees built in to pricing, and 2) use of a combination of public domain procedures for assay construction (e.g. ink-jet deposition of reagents and available nitrocellulose assay membranes) to avoid the necessity for technology in-licensing. From a technical perspective we need to demonstrate that our "avidity" anti-CD4+ capture reagent displays a dissociation constant for CD4+ expressing T cells approaching zero and is thereby capable of CD4+ cell capture. The total HIV positive patient population worldwide is in excess 40 million. The vast majority of individuals living with this disease are in resource poor environments where conventional CD4 enumeration is both too expensive to perform and technically challenging, due to a paucity of trained personnel. Treatment efforts currently underway, such as the World Health Organizations "3 by 5" Initiative, will be providing access to HAART (e.g. highly active anti-retroviral therapy) to millions of patients in these areas of the world over the next several years. It is in such resource poor environments where CD4 counts are arguably the most important to perform. Current costs and assay complexities limit this. An accurate CD4 count can be employed: to facilitate AIDS surveillance; to monitor the rate of progression to AIDS, to define when therapy is required to prevent opportunistic infections, to place drug-naive patients into cohorts prior to therapy, and to monitor the effects of anti-retroviral therapy. It is currently recommended that a CD4 assay is performed on every HIV infected individual every 3-6 months and more frequently depending on circumstance. The assay described here is intended to answer this need, both from the standpoint of addressing the technical difficulties and the cost requirements. It is currently believed that lack of such an assay leads to unneeded death due to opportunistic infections. The type of rapid assay being developed under this phase I proposal, if successful, could have a dramatic impact on the ability to detect (and thereby enable appropriate treatment) opportunistic infections in the developing world for HIV infected individuals on anti-retroviral therapy.