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.
The proposed project describes the very great improvement of the cheap, common 'pregnancy test' format for diagnostics through the use of enzyme-decorated, harmless viral nanoparticles. Results to date suggest this system to be 100-fold superior in sensitivity over current clinical diagnostic assays; we plan to investigate why and move the technology toward widespread use. Great sensitivity, low cost, and ease of use make it highly relevant for the earlier diagnosis of diseases in an effort to advance the nation's capacity to protect and improve public health.
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