A lateral flow CD4 counting assay for resource-poor regions

描述(由申请人提供)：目前对艾滋病毒感染者的CD4+细胞计数的常规测量的需求是巨大的。目前，CD4+计数的成本是一个主要问题，尤其是在感染率最高的资源匮乏环境中。在资源匮乏的环境中，获得准确的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、细胞分类器、平板阅读器)，这些设备需要受控的环境(在高湿度和非标准温度环境下的机器维护是有问题的)。这项提议旨在开发一种快速的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”)和一种专利和廉价的方法来创建抗CD4+“亲和力”捕获试剂。这种分析将能够在资源匮乏的环境中进行，不需要昂贵的设备，对于环境变量是健壮的，并且可以由不熟练的人员执行和解释。我们建议，可以制造具有这些属性的准确的CD4+计数检测，最终用户每次检测的成本不到1.00美元。这一化验成本估算部分基于两个假设：1)化验的所有试剂都将在内部开发，以规避定价中内置的试剂许可费，以及2)使用公共领域程序的组合来构建化验(例如，试剂的喷墨沉积和可用的硝化纤维化验膜)，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“亲和力”抗CD4+捕获试剂对表达CD4+的T细胞显示出接近零的解离常数，从而能够捕获CD4+细胞。全世界艾滋病毒阳性患者的总人数超过4000万。绝大多数患有这种疾病的人生活在资源匮乏的环境中，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努力，如世界卫生组织“3×5”倡议，将在未来几年为世界这些地区的数百万患者提供接受HAART(例如，高度有效的抗逆转录病毒疗法)的机会。正是在这种资源匮乏的环境中，可以说，CD4计数是最重要的。目前的成本和分析的复杂性限制了这一点。可以使用准确的CD4计数：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；在治疗前将未服用药物的患者分成队列，以及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一次CD4检测，并根据情况更频繁地进行检测。从解决技术困难和成本要求的角度来看，这里描述的化验方法旨在满足这一需求。目前认为，由于机会性感染，缺乏这样的检测方法会导致不必要的死亡。根据这一第一阶段提案正在开发的这类快速化验如果成功，可能会对发展中国家接受抗逆转录病毒治疗的艾滋病毒感染者发现机会性感染(从而能够进行适当治疗)的能力产生重大影响。

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.