title: Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses.
abstract: Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)-named NJT-R3-A2 and NJT-R3-A3-that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics.
authors.affiliation: Interdepartmental Program in Translational Biology & Molecular Medicine, Baylor College of Medicine, Houston, Texas, United States of America.|Department of Pharmacology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Hurwitz
authors.affiliation: Department of Pharmacology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Wanzhi
authors.initials: W
authors.lastname: Huang
authors.affiliation: Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Baijun
authors.initials: B
authors.lastname: Kou
authors.affiliation: Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.|Department of Medicine, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Estes
authors.affiliation: Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.|Department of Medicine, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Atmar
authors.affiliation: Department of Pharmacology, Baylor College of Medicine, Houston, Texas, United States of America.|Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Timothy
authors.initials: T
authors.lastname: Palzkill
mesh_terms: D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody; D017250:Caliciviridae Infections / Q000276:immunology* / Q000821:virology; D006801:Humans; D029322:Norovirus / Q000145:classification / Q000276:immunology* / Q000378:metabolism*; D019151:Peptide Library; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000378:metabolism*; D014771:Virion / Q000276:immunology* / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D019151:Peptide Library; D057127:Single-Chain Antibodies