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 enzymelinked 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.

Porcine epidemic diarrhea: A retrospect from Europe and matters of debate

“…So far, only discrete antigenic relationship involving the N protein but without any cross protection was detected with some of the other animal members of the genus alphacoronavirus such as feline infectious peritonitis virus (FIPV), TGEV, porcine respiratory coronavirus (PRCV), canine coronavirus (CCoV) and mink coronavirus (MCV). By the use of monoclonal antibodies to the N proteins of the human alphacoronaviruses NL63 and 229E, no cross reactivity was detected with PEDV (Sastre et al, 2011). The only alphacoronavirus in which also the M proteins cross reacts with PEDV is MCV (Have et al, 1992).…”

Influences of cyclosporin A and non-immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229E replication

“…Mouse antibody 1H11 (1:20,000) recognizing HCoV-229E N-protein was obtained from INGENASA, Spain (Sastre et al, 2011). Anti-Lamin A (A303-433A, [1:20,000]), anti-PPIA (ab3563, [1:500]) and anti-PPIB (PA1-027A, [1:800]) were purchased from Biomol, Abcam and Ther-moFisher, respectively.…”

Human coronavirus NL63 replication is cyclophilin A-dependent and inhibited by non-immunosuppressive cyclosporine A-derivatives including Alisporivir

“…Mouse antibody 1H11 (1:20,000) recognizing HCoV-NL63 Nprotein was obtained from INGENASA, Spain (Sastre et al, 2011). Anti-Lamin A (1:20,000) was purchased from Biomol, Hamburg, Germany.…”