Development of a recombinant truncated nucleocapsid protein based immunoassay for detection of antibodies against human coronavirus OC43

Abstract

Human coronaviruses are one of the main causes of upper respiratory tract infections in humans. While more often responsible for mild illness, they have been associated with illnesses that require hospitalization. In this study, an assay for one of the human coronaviruses, OC43, was developed using a truncated recombinant nucleocapsid (N) protein antigen in an enzyme immunosorbent assay (ELISA) and evaluated using serum collected from HCoV-OC43-infected patients, healthy adults, and patients with other respiratory virus infections. Results showed that the diagnostic sensitivity and specificity of the assay were 90.9% (10/11) and 82.9% (39/47), respectively. To evaluate the clinical utility of the ELISA, serum samples collected from patients during an outbreak of HCoV-OC43 infection and previously identified as positive by HCoV-OC43 whole N ELISA were screened resulting in 100% diagnosis agreement between the testing methods. These results suggest that this assay offers a reliable method to detect HCoV-OC43 infection and may be a useful tool in coronavirus seroepidemiological studies.

Fig. 1

Analysis of the purified recombinant truncated HCoV-OC43 nucleocapsid (N) proteins by SDS-PAGE (A) and Western blot (B). Proteins were separated by SDS-PAGE and analyzed by immunoblotting using anti-His antibody. Lane 1: HCoV-OC43 N1 truncated protein (aa1–119); lane 2: HCoV-OC43 N2 protein (aa120–332); lane 3: HCoV-OC43 N3 protein (aa333–448). Expected band sizes: HCoV-OC43 N1 (aa1–119) 17 kDa, HCoV-OC43 N2 (aa120–332) 25 kDa, and HCoV-OC43 N3 (aa333–448) 17.5 kDa.

Fig. 2

Specificity and cross-reactivity of patient sera samples for truncated human coronavirus OC43 (HCoV-OC43) N protein antigen candidates. (A) Sera samples acute HCoV-OC43 (n = 15), convalescent OC43 (n = 11), HCoV-229E (n = 27), and SARS-CoV serum samples (n = 20) were screened for reactivity to HCoV-OC43 N1, N2 and N3 truncated proteins. Error bars represent the standard error of the mean. (B) Sera from HCoV-229E patients (n = 27), and SARS-CoV patients (n = 18), were screened for cross-reactivity against HCoV-OC43 whole nucleocapsid and HCoV-OC43 N3 proteins by ELISA. Specimens were run in duplicates and the optical densities at 405 nm (OD₄₀₅) with a 490 nm reference filter for each samples are shown. The line represents the median OD values for each sample set.

Fig. 3

Cut-off analysis for Human coronavirus OC43 (HCoV-OC43) N3 based ELISA and comparison with HCoV-OC43 whole N based ELISA. (A) The association between sensitivity (Se) and specificity (Sp) and OD value cut-offs for the HCoV-OC43 N3 based ELISA was determined (B) Comparison of average optical densities (OD₄₀₅) for acute (n = 15) and convalescent (n = 11) HCoV-OC43 samples against HCoV-OC43 N3 and whole HCoV-OC43 N based ELISAs.

Fig. 4

Scatter chart of absorbance values with representative sera from (A) HCoV-OC43 (OC43), HCoV-229E (229E), SARS patients and (B) healthy U.S. blood donors (NHS) for IgG antibodies tested by HCoV-OC43 N3 protein based ELISA. Results are plotted as optical density values at 405 nm with a 490-nm reference filter. The black line indicates the cutoff value of 0.161 as determined by ROC analysis.

Fig. 5

Graph of absorbance values with acute (n = 8) and convalescent (n = 9) specimens from 10 patients collected during an HCoV-OC43 outbreak for IgG antibodies to recombinant HCoV-OC43 N3 protein. Results are plotted as optical density values at 405 nm with a 490-nm reference filter. The black line indicates the cut-off value of 0.161.