Viral nanoparticles as ultrasensitive reporters in lateral-flow assays

 描述(由申请人提供)：免疫层析侧向流动分析(LFA)是一种成熟、廉价的临床点诊断和分析方法，已被证明适用于各种样本类型和各种环境下的初步测试。侧向流动分析被广泛使用，并且可以说是能够满足特定需求的首选方法，因为它们易于使用且成本较低。然而，侧向流动分析的检测极限比更复杂的实验室方法，如酶联免疫吸附试验和聚合酶链式反应更差。我们的初步结果表明，使用抗体和报告酶修饰的噬菌体颗粒作为LFA报告颗粒，可以将LFA的检测下限提高100倍或更多，甚至比使用酶修饰的纳米颗粒更多。这项计划工作的意义在于，这种方法将在不牺牲特异性的情况下提高诊断的敏感性，并通过先发制人的更复杂的方法，减少诊断成本和时间。在初步研究中，我们已经证明，使用功能化的高表面积丝状噬菌体纳米颗粒作为亲和剂的LFA分析可以非常成功，即使在抗体方便地共价偶联的情况下也是如此，即无需克隆。我们的假设是，用酶修饰的丝状噬菌体纳米粒进行侧向流动分析可以大大提高检测限，而不是传统的胶体金LFA，并且接近ELISA。为了验证这一假设，我们将研究超敏感性的机制，并比较使用噬菌体和传统的侧向流动试验和ELISA对各种模型分析物的检测。这项工作将验证拟议的新的护理点诊断平台，并可能产生普遍有用的原则，以提高LFA的敏感性。通过视频显微镜，我们将观察不同噬菌体在LFA基质中的传输和结合，从而更深入地了解噬菌体形状和大小以及支架形态对LFA性能的影响(其他棒状颗粒垂直于受限几何形状中的流动，这可能促进在LFA中的捕获)。然后，我们将优化LFA的材料和稳定性，并使用模型分析物确定噬菌体LFA的检测极限。在样本中加入浓度降低的鸡蛋溶菌酶和MS2噬菌体，并使用抗体和过氧化物酶双重标记的M13噬菌体作为报告颗粒进行分析，并与使用相同抗体的ELISA和常规胶体金LFA进行比较。我们最终将使噬菌体免疫层析分析方法用于检测血液中的分析物，使用血液过滤器和洗涤剂钝化，并将噬菌体外壳蛋白与聚乙二醇接枝。在横向流动分析中，使用丝状噬菌体作为多酶报告的支架构成了一种新的方法，可以极大地提高检测限，对所有护理点分析物检测领域都有直接影响。

 DESCRIPTION (provided by applicant): Immuno-chromatographic lateral flow assays (LFAs) are a well-established, inexpensive point-of-care diagnostic and analytical method with proven utility for the primary testing of a diverse range of sample types and in a wide variety of setting. Lateral flow assays are very widely used, and arguably are the preferred approach when they are capable of meeting a given need because of their ease of use and low cost. The limits of detection of lateral flow assays are worse, however, than those of more elaborate laboratory methods such as ELISA and PCR. Our preliminary results suggest that the use of phage particles decorated with antibodies and reporter enzymes as LFA reporter particles can improve LFA limits of detection by 100-fold or more, even much more than the use of enzyme-decorated nanoparticles. The significance of the planned work is that this method will improve diagnostic sensitivity without sacrificing specificity and, by pre-empting more elaborate methods, will reduce cost and time to diagnosis. In preliminary studies we have shown that an LFA assay with functionalized high-surface area filamentous phage nanoparticles as the affinity agent can be very successful, even when antibodies are conveniently covalently coupled, i.e., without cloning. Our hypothesis is that lateral flow assays with enzyme-modified filamentous phage nanoparticles can greatly improve limits of detection over conventional colloidal-gold LFAs, and approach ELISAs. To test this hypothesis we will investigate the mechanisms of ultrasensitivity, and compare the detection of various model analytes using phage and conventional lateral flow assays and ELISA. This work will validate the proposed new point-of-care diagnostic platform, and may also produce generally useful principles for improving LFA sensitivity. By video microscopy we will observe transport and binding of varied phage in LFA matrices and acquire a deeper understanding of the effects of phage shape and size and scaffold morphology on performance in LFAs (other rodlike particles orient perpendicular to flow in confined geometries; this may promote capture in LFA). We will then optimize LFA materials and stability and determine the limit of detection for a phage-LFA using model analytes. Samples will be spiked with decreasing concentrations of hen egg lysozyme and the phage MS2 and analyzed using antibody- and peroxidase-doubly-labeled M13 bacteriophage as reporter particles, with comparisons to ELISA and conventional colloidal-gold LFA using the same antibodies. We will finally adapt the phage immuno-chromatographic assay to detect analytes in blood, with blood filters and passivation by detergents and the grafting of the phage coat proteins with PEG. The use of filamentous phage as a scaffold for multiple enzyme reporters in lateral flow assays constitutes a novel approach that can allow a great improvement in detection limits, with immediate impact in all areas of point-of-care analyte detection.