Profile of Antibodies to the Nucleocapsid Protein of the Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus in Probable SARS Patients

Abstract

Profiles of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (SARS)-associated coronavirus in 445 probable SARS patients and 3,749 healthy people or non-SARS patients were analyzed by antigen-capturing enzyme-linked immunosorbent assay. Antinucleocapsid antibodies were elucidated in 17.5% of the probable SARS patients 1 to 7 days after the onset of symptoms and in 80% of the patients 8 to 14 days after the onset. About 90% of the probable SARS patients were positive 15 or more days after illness. Antibody titers increased up to 70 days, and high antibody titers were maintained at least for another 3 months. Of the healthy people and non-SARS patients, only seven (0.187%) were weakly positive.The novel severe acute respiratory syndrome (SARS)-associated coronavirus (CoV) has been identified as the etiologic agent of SARS (1,3,5). It has been demonstrated that, at least in early responses, the antibodies to the nucleocapsid protein (N protein) predominate, as assayed by Western blotting and proteomic analysis. To understand the humoral immunity to the N protein of SARS CoV and the possibility of using the N protein in SARS diagnosis, antibodies to the N protein from 445 patients who probably had SARS, as diagnosed on the basis of World Health Organization criteria, from four hospitals were analyzed by an antigen-capturing enzyme-linked immunosorbent assay in which recombinant SARS N protein was used as the antigen (6). The method is briefly described as follows. The N-encoding gene of SARS CoV was cloned into T7 promoter-based prokaryotic expression vector pET22b (Novagen), and the resulting recombinant plasmid (pMG-N) was then transformed into Escherichia coli BL21a(DE3). The recombinant N protein was expressed in E. coli by induction with isopropyl-␤-D-thiogalactopyranoside (IPTG; Sigma, St. Louis, Mo.) at 0.5 mmol/liter and purified by S-Sepharose Fast Flow ion-exchange chromatography, followed by gel filtration with Superdex 200 (Amersham Pharmacia) to a purity of Ͼ97% as determined by laser densitometry of a silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. The purified N protein was diluted to 1 g/ml with 50 mM carbonate buffer (pH 9.6) and used to coat the wells of 96-well microplates at 4°C overnight, followed by blocking with 5% fetal bovine serum for 4 h at room temperature. In addition, N protein was conjugated to horseradish peroxidase (Sigma). An antigen-capturing enzyme-linked immunosorbent assay was established for the detection of anti-N protein antibody present in sera. A 100-l volume of serum was added to the well coated with recombinant N protein, and the plate was incubated at 37°C for 30 min and then washed five times with phosphatebuffered saline containing 0.05% Tween 20. A 10-l volume of labeled antigen was added, and the plate was incubated for another 30 min and washed as already described, and then 100 l of TMB substrate solution (0.1 mg of tetramethylbenzidine hydrochloride/ml, 0.01% H 2 O 2 in 0.1 M acetate buffer, pH 5.8) was ...

Use of the COOH Portion of the Nucleocapsid Protein in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay for Specific and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus

“…Since the specific antibodies to nucleocapsid (N) protein are most abundant in the sera from SARS patients (5), several groups have developed some recombinant N protein-based ELISAs. In general, these new assays are more specific and sensitive than the ELISA based on whole-virus lysate, but some false-positive results still occurred with sera from non-SARS patients or healthy people, even with sera collected several years before the SARS outbreak (3,7,8,(10)(11)(12).…”

“…Since the specific antibodies to nucleocapsid (N) protein are most abundant in the sera from SARS patients (5), several groups have developed some recombinant N protein-based ELISAs. In general, these new assays are more specific and sensitive than the ELISA based on whole-virus lysate, but some false-positive results still occurred with sera from non-SARS patients or healthy people, even with sera collected several years before the SARS outbreak (3,7,8,(10)(11)(12).Since the N proteins of the known coronaviruses are relatively conserved, the expressed N protein of the SARS-CoV in Escherichia coli cross-reacts with polyclonal antisera of some known animal coronaviruses in antigenic group I, including transmissible gastroenteritis virus, feline infectious peritonitis virus, and canine coronavirus (9), which raised concerns of potential false positives when the recombinant N protein of the SARS-CoV, whole-virus antigen extracts, or virus-infected cells are used as reagents. However, this concern is very minimal, since the two known human coronaviruses (strains 229E and OC43) do not cause severe clinical diseases and we still do not have data about the prevalence of the antibodies to other coronaviruses or relevant microorganisms in human populations (4,6,8).…”

Recombinant Protein-Based Assays for Detection of Antibodies to Severe Acute Respiratory Syndrome Coronavirus Spike and Nucleocapsid Proteins

“…Although very few serum specimens from unexposed persons (Ͻ1.5%) tested positive for SARS-CoV infection, the potential for cross-reactivity between SARSCoV and other coronaviruses, including the known human coronaviruses HCoV-OC43, HCoV-229E, and recently identified HKU1 and NL63, remains a concern (12,13,22). Whether these positive results are due to nonspecific reactivity to the recombinant SARS N protein or to cross-reactivity to other human CoVs requires further study.…”

“…Previous studies have demonstrated the utility of anti-N and anti-S proteins in the diagnosis of SARS-CoV infections (2,5,12,21). In this study, we describe the evaluation and comparison of recombinant spike and nucleocapsid enzyme-linked immunosorbent assays (ELISAs) for specifically detecting SARS-CoV infection.…”

“…Persistent levels of SARS-CoV IgG have been detected in SARS cases for several months and up to 2 years after disease onset (4,11,12,19,21). In this study, serum samples from 48 SARS patients from Vietnam and the United States were collected 221 to 735 days (44 from days 221 to 250; 4 from days 633 to 735) after the onset of illness and tested for the presence of SARS-CoV-and N-and S-protein-specific IgG by ELISA.…”