title: Differentiation between human coronaviruses NL63 and 229E using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies.
abstract: Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in an Escherichia coli system and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections.
authors.affiliation: Inmunología y Genética Aplicada S.A. (INGENASA), Hnos. García Noblejas 41, 28037 Madrid, Spain. psastre@ingenasa.es
authors.forename: Patricia
authors.initials: P
authors.lastname: Sastre
authors.forename: Ronald
authors.initials: R
authors.lastname: Dijkman
authors.forename: Ana
authors.initials: A
authors.lastname: Camuñas
authors.forename: Tamara
authors.initials: T
authors.lastname: Ruiz
authors.forename: Maarten F
authors.initials: MF
authors.lastname: Jebbink
authors.forename: Lia
authors.initials: L
authors.lastname: van der Hoek
authors.forename: Carmen
authors.initials: C
authors.lastname: Vela
authors.forename: Paloma
authors.initials: P
authors.lastname: Rueda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood / Q000276:immunology; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D000918:Antibody Specificity; D028941:Coronavirus 229E, Human / Q000145:classification* / Q000276:immunology; D018352:Coronavirus Infections / Q000175:diagnosis* / Q000276:immunology / Q000382:microbiology; D058957:Coronavirus NL63, Human / Q000145:classification* / Q000276:immunology; D000086462:Coronavirus Nucleocapsid Proteins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology*; D013045:Species Specificity
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins