Browsing by Author "Adhikari, Meena"
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Item Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays(Analytical Chemistry, 2016-12) Adhikari, Meena; Strych, Ulrich; Kim, Jinsu; Goux, Heather J.; Dhamane, Sagar; Poongavanam, Mohan-Vivekanandan; Hagstrom, Anna E. V.; Kourentzi, Katerina D.; Conrad, Jacinta C.; Willson, Richard C.We introduce the modification of bacteriophage particles with aptamers for use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin朾iotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ?100 times lower than those of previously reported IgE assays.Item Detection of Viruses By Counting Single Fluorescent Genetically Biotinylated Reporter Immunophage Using a Lateral Flow Assay(ACS Appl Mater Interfaces, 2016-02) Kim, Jinsu; Adhikari, Meena; Dhamane, Sagar; Hagstrom, Anna E. V.; Kourentzi, Katerina D.; Strych, Ulrich; Willson, Richard C.; Conrad, Jacinta C.We demonstrated a lateral flow immunoassay (LFA) for detection of viruses using fluorescently labeled M13 bacteriophage as reporters and single-reporter counting as the readout. AviTag-biotinylated M13 phage were functionalized with antibodies using avidin朾iotin conjugation and fluorescently labeled with AlexaFluor 555. Individual phage bound to target viruses (here MS2 as a model) captured on an LFA membrane strip were imaged using epi-fluorescence microscopy. Using automated image processing, we counted the number of bound phage in micrographs as a function of target concentration. The resultant assay was more sensitive than enzyme-linked immunosorbent assays and traditional colloidal-gold nanoparticle LFAs for direct detection of viruses.Item Sensitive Detection of Norovirus Using Phage Nanoparticle Reporters in Lateral-Flow Assay(PLoS ONE, 2015-05) Hagstrom, Anna E. V.; Garvey, Gavin; Paterson, Andrew S.; Dhamane, Sagar; Adhikari, Meena; Estes, Mary K.; Strych, Ulrich; Kourentzi, Katerina D.; Atmar, Robert L.; Willson, Richard C.Noroviruses are recognized worldwide as the principal cause of acute, non-bacterial gastroenteritis, resulting in 19-21 million cases of disease every year in the United States. Noroviruses have a very low infectious dose, a short incubation period, high resistance to traditional disinfection techniques and multiple modes of transmission, making early, point-of-care detection essential for controlling the spread of the disease. The traditional diagnostic tools, electron microscopy, RT-PCR and ELISA require sophisticated and expensive instrumentation, and are considered too laborious and slow to be useful during severe outbreaks. In this paper we describe the development of a new, rapid and sensitive lateral-flow assay using labeled phage particles for the detection of the prototypical norovirus GI.1 (Norwalk), with a limit of detection of 107 virus-like particles per mL, one hundred-fold lower than a conventional gold nanoparticle lateral-flow assay using the same antibody pair.