A sensitive, low-cost, fluorescence assay for determination of HIV p24 in infants

描述（由申请人提供）：每年有150万名儿童由感染艾滋病毒的母亲所生，导致35万例艾滋病毒垂直传播给新生儿。早期检测后及时进行抗逆转录病毒治疗（ART）可以挽救生命，但缺乏婴儿护理点（POC）检测会导致治疗延迟或缺乏治疗。我们的方法将提供一种低成本但定量和灵敏的侧流免疫测定（LFIA）测试来分析和定量HIV p24的存在。 与现有的快速检测相比，荧光染料将用于提高检测限。智能手机用于成像、分析和自动报告。长斯托克斯位移染料，R-藻红蛋白（PE）将被用作荧光报告，无论是单独或捆绑成一个集群。PE的光学性质允许使用廉价的照明设备; PE的最小非特异性结合导致具有宽动态范围的灵敏检测。通过设计用于解离靶蛋白免疫复合物以改善测试试剂的接近性的集成测试功能，将实现灵敏度的额外增益。 在第一阶段，我们将：1）优化抗HIV p24抗体上的PE报告子（簇）; 2）开发智能手机应用程序以允许自动化分析; 3）开发LFIA条上的“解离区”以帮助分析复合的p24; 4）开发使用对照线作为侧流条上的定量标准以允许p24的绝对定量; 5）设计、制造和测试低成本荧光系统的原型，所述荧光系统的材料成本低于35美元。 6)通过将结果与使用加标重组HIV p24的热灭活血清的市售ELISA和LFIA检测进行比较，验证p24 LFIA。 这些调查将证明我们的系统检测和分析HIV p24的性能优势。对于第二阶段，我们希望测试真实的患者样本。

DESCRIPTION (provided by applicant): Each year, 1.5 million children are born to mothers living with HIV resulting in vertical transmission of HIV to newborns in 350,000 cases. Early testing followed by prompt anti-retroviral therapy (ART) is known to save lives, yet lack of point-of-care (POC) tests for infants results in delays or absence of therapy. Our approach will provide a low-cost yet quantitative and sensitive lateral flow immunoassay (LFIA) test to analyze and quantitate the presence of HIV p24. Fluorescent dyes will be used to improve the limit of detection compared to existing rapid tests. A smart phone is utilized for imaging, analysis and automated reporting. A long Stokes shift dye, R-phycoerythrin (PE) will be used as the fluorescent reporter, either singly or bundled into a cluster. The optical properties of PE allow the use of an inexpensive illumination device; the minimal non-specific binding of PE results in sensitive detection with a wide dynamic range. Additional gains in sensitivity will be achieved through an integrated test feature designed to dissociate target protein immune-complexes for improved access by the test reagents. In Phase I we will: 1) Optimize the PE reporter (cluster) on anti-HIV p24 antibody; 2) Develop a smartphone application to allow automated analysis; 3) Develop an on-LFIA strip "zone of dissociation" to aid in analysis of complexed p24; 4) Develop the use of the control line as a quantification standard on the lateral flow strip to allow absolut quantitation of p24; 5) Design, fabricate and test a prototype of a low cost fluorescence system designed with a materials cost less than $35. 6) Validate the p24 LFIAs by comparing results with commercially available ELISA and LFIA tests using heat inactivated serum spiked with recombinant HIV p24. These investigations will demonstrate the performance advantage of our system for detection and analysis of HIV p24. For phase II we hope to test authentic patient samples.