title: Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus.
abstract: Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. These viruses are usually detected by molecular methods, including reverse transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Human noroviruses are genetically and antigenically diverse, with two main genogroups that are further subdivided into over 40 different genotypes. During the past decade, genogroup 2 genotype 4 (GII.4) has dominated in most countries, but recently, viruses belonging to GII.17 have increased in prevalence in a number of countries. A number of commercially available ELISAs and lateral flow immunoassays were found to have lower sensitivities to the GII.17 viruses, indicating that the antibodies used in these methods may not have a high level of cross-reactivity. In this study, we developed a rapid Nanobody-based lateral flow immunoassay (Nano-immunochromatography [Nano-IC]) for the detection of human norovirus in clinical specimens. The Nano-IC assay detected virions from two GII.4 norovirus clusters, which included the current dominant strain and a novel variant strain. The Nano-IC method had a sensitivity of 80% and specificity of 86% for outbreak specimens. Norovirus virus-like particles (VLPs) representing four genotypes (GII.4, GII.10, GII.12, and GII.17) could be detected by this method, demonstrating the potential in clinical screening. However, further modifications to the Nano-IC method are needed in order to improve this sensitivity, which may be achieved by the addition of other broadly reactive Nanobodies to the system.IMPORTANCEWe previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical setting, the Nano-IC assay had sensitivities equivalent to other commercially available lateral flow systems. The Nano-IC method was capable of producing results in ~5 min, which makes this method useful in settings that require rapid diagnosis, such as cruise ship outbreaks and elder care facilities. The Nano-IC assay has several advantages over antibody-based IC methods: for example, Nanobodies can be readily produced in large quantities, they are generally more stable than conventional antibodies, and the Nanobody binding sites can be easily obtained by X-ray crystallography.
authors.affiliation: Schaller Research Group at the University of Heidelberg and the DKFZ, Heidelberg, Germany; Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Sylvie Y
authors.initials: SY
authors.lastname: Doerflinger
authors.affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany; Child and Adolescent Medicine, General Pediatrics, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Tabatabai
authors.affiliation: Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Schnitzler
authors.affiliation: R-Biopharm AG, Darmstadt, Germany.
authors.forename: Carlo
authors.initials: C
authors.lastname: Farah
authors.affiliation: R-Biopharm AG, Darmstadt, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Rameil
authors.affiliation: R-Biopharm AG, Darmstadt, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Sander
authors.affiliation: Schaller Research Group at the University of Heidelberg and the DKFZ, Heidelberg, Germany; Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Koromyslova
authors.affiliation: Schaller Research Group at the University of Heidelberg and the DKFZ, Heidelberg, Germany; Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Grant S
authors.initials: GS
authors.lastname: Hansman
keywords: Nanobody; lateral flow assay; norovirus