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.

FIG. 1.

Immunoblot analysis of HCoV N protein. (A) Reactivity with an MAb specific for HCoV-NL63 (MAb 2D4); (B) reactivity with an MAb specific for HCoV-229E (MAb 1E7); (C) reactivity with an MAb that recognizes both viruses (MAb 5D5). Lanes 1, N protein of HCoV-NL63; lanes 2, N protein of HCoV-229E; lane 0, molecular weight markers (numbers on the left are molecular weights, in thousands).

FIG. 2.

Reactivity of MAbs with the N protein and the C-terminal half of the N proteins of HCoVs. (A) Reactivity of MAbs specific for HCoV-NL63 (B); reactivity of MAbs specific for HCoV-229E; (C) reactivity of MAbs that recognize both viruses. ELISA plates were coated with the recombinant N protein of HCoV-NL63 (black bars) or HCoV-229E (white bars) or the corresponding C-terminal fragments (dotted and dashed bars, respectively). The y axis represents the absorbance readings (OD₄₅₀) obtained in the indirect ELISA.

FIG. 3.

Detection of viral antigen in DAS-ELISA. (A) Detection of HCoV-NL63; (B) detection of HCoV-229E. Purified MAb 1E8 was used as the capture antibody. Serial dilutions of virus-infected cell lysates were added to the plates, and the viral antigen was detected by using a species-specific peroxidase-conjugated MAb (MAbs 2D4-PO and 1E7-PO for HCoV-NL63 and HCoV-229E detection, respectively). The absorbance at 450 nm was plotted against the viral titer, expressed as the numbers of TCID₅₀s/ml.

FIG. 4.

Alignment of the amino acid sequences of the C-terminal half of the nucleocapsid proteins of various HCoVs. Highly conserved regions between the four aligned sequences are in shaded boxes. Sequences used for the alignment included those of the following viruses: HCoV-229E (GenBank accession no. NC_002645), HCoV-NL63 (GenBank accession no. NC_005831), HCoV-HKU1 (GenBank accession no. NC_006577), and HCoV-OC43 (GenBank accession no. NC_005147). The N protein of HCoV-229E was considered the reference sequence (aa 246 to 286), and the alignment was performed with DS Gene (version 1.5) software.