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Developing a Primary Concentration Method Using Antibody Fragments to Capture Enteric Viruses in Source Waters

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Title: Developing a Primary Concentration Method Using Antibody Fragments to Capture Enteric Viruses in Source Waters
Author: Mahrous, Nahed
Program: Environmental Sciences
Advisor: Habash, Marc
Abstract: Human enteric viruses (EVs) are the commonest causes of waterborne gastroenteritis. Principal challenges to monitoring EVs from water sources include filtering large volumes of water and low recoveries, which can negatively affect downstream applications for detection. Bioactive paper is a low-cost and easy-to-use paper-based sensor that can rapidly and specifically capture and detect pathogens from a sample. We developed antibody-based technologies using cellulose filters and magnetic beads for the capture and detection of EVs from water samples. As a proof-of-concept, RV group A strains, published monomeric single domain antibodies (sdAbs, RV-2KD1 and RV-3B2) and full-length IgG (RV-IgG 26) specific to the RV capsid protein VP6 were selected. For the antibody-cellulose filter technology, the sdAbs were linked to a cellulose binding module (CBM2a) for attachment to cellulose filter paper. For the antibody-bead technology, the sdAbs and RV-IgG 26 were chemically conjugated to magnetic beads. Western blot and enzyme-linked immunosorbent assay (ELISA) confirmed all RV-mAbs specifically bound RV strains from human and bovine sources. ELISA indicated that the N-terminal linking of 2KD1 to CBM2a was shown to be the most effective. RV recovery by both technologies from spiked tap and river water was analyzed via quantitative PCR and scanning electron microscopy. RV recoveries of 27.44 ± 22.93% and 26.72 ± 19.66% with RV alone and 29.18 ± 2.38% and 32.66 ± 8.74% in competition with adenovirus from tap and river waters, respectively, were obtained using the filter-based technology. RV recoveries from river water in competition with adenovirus were 1.85 ± 1.46% and 0.87 ± 0.4% using RV-IgG 26- and RV-2KD1-coupled beads, respectively, using the magnetic bead-based technology. SEM confirmed the attachment of RV on the antibody-linked filter and the beads. Both technologies yielded non-specific binding which is a well-known challenge with antibody-based assays (e.g., ELISA). More optimization steps are needed to improve the capturing of the virus by these developed technologies and minimize non-specific binding. The successful development of a filter-based capture technology for the detection of EVs will provide a critical tool for rapid and cost-effective routine monitoring of critical human pathogens to inform water safety and treatment.
URI: http://hdl.handle.net/10214/17471
Date: 2019-09
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