Viral nanoparticles as ultrasensitive reporters in lateral-flow assays

 描述（由申请方提供）：免疫色谱侧流检测试剂盒（LFA）是一种成熟、廉价的床旁诊断和分析方法，经证明可用于各种样本类型和各种环境的初步检测。侧向流测定法被非常广泛地使用，并且当它们能够满足给定的需要时，由于它们的易用性和低成本，可以说是优选的方法。然而，侧流检测的检测极限比ELISA和PCR等更精细的实验室方法更差。我们的初步研究结果表明，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检测限提高100倍或更多，甚至比使用酶修饰的纳米颗粒更高。计划工作的意义在于，这种方法将提高诊断灵敏度，而不牺牲特异性，并通过抢先采用更复杂的方法，降低诊断成本和时间。在初步研究中，我们已经表明，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测定可以非常成功，即使当抗体方便地共价偶联时，即，没有克隆。我们的假设是，酶修饰的丝状噬菌体纳米颗粒的侧流检测可以大大提高检测限超过传统的胶体金LFA，并接近ELISA。为了验证这一假设，我们将研究超灵敏度的机制，并比较使用噬菌体和传统的侧流分析和ELISA检测各种模型分析物。这项工作将验证所提出的新的即时诊断平台，也可能产生普遍有用的原则，提高LFA的灵敏度。通过视频显微镜，我们将观察不同的噬菌体在LFA基质中的运输和结合，并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（其他棒状颗粒在受限的几何形状中垂直于流动方向;这可能会促进LFA中的捕获）。然后，我们将优化LFA材料和稳定性，并使用模型分析物确定噬菌体-LFA的检测限。样品将加标浓度递减的鸡蛋溶菌酶和噬菌体MS 2，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，与ELISA和使用相同抗体的常规胶体金LFA进行比较。我们将最终适应噬菌体免疫层析测定检测血液中的分析物，血液过滤器和钝化的洗涤剂和接枝的噬菌体外壳蛋白与PEG。使用丝状噬菌体作为支架的多个酶的报告在侧流测定构成了一种新的方法，可以允许极大地提高检测限，在所有领域的即时护理分析物检测的直接影响。

 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.