Diagnosis of Severe Acute Respiratory Syndrome (SARS) by Detection of SARS Coronavirus Nucleocapsid Antibodies in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay

Abstract

Recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein was employed to establish an antigen-capturing enzyme-linked immunosorbent assay (ELISA). Antinucleocapsid protein antibodies could be detected in 68.4% of probable SARS patients 6 to 10 days after illness and in 89.6% of the patients 11 to 61 days after illness. No false-positive results were observed in 20 non-SARS fever patients, 24 non-SARS respiratory illness patients, and 20 health care workers. Among 940 other non-SARS clinical serum samples, only 1 was found to be weakly positive. This method provides a new, sensitive, and specific approach for SARS diagnosis.An accurate, rapid, and cost-effective laboratory etiologic method is of great importance for the diagnosis of severe acute respiratory syndrome (SARS). The isolation of the virus (4) led to the development of some specific diagnostic techniques, including indirect fluorescent-antibody detection, indirect enzyme-linked immunosorbent assay (ELISA) using virus lysates as antigen, and reverse transcription PCR for the detection of the SARS coronavirus (SARS CoV) genome (5). However, as observed in the clinical practices of China and shown in this paper, indirect ELISA gave about 2% false-positive results among healthy people; SARS-CoV infection could be confirmed only if seroconversion from negative to positive status was observed.Antigen-capturing ELISA is a superior method to indirect immunoassay because of its high specificity and sensitivity. The basis of the assay is that antibodies are at least bivalent, i.e., one valence is used in attaching the antibody to the immobilized antigen, leaving the other(s) free to bind to the labeled antigen. Both capture and detection of the target antibody depend on its specificity toward the antigen, so if the antigen is correctly chosen and purified, the assay can be made very specific. And principally, all types of antibodies (immunoglobulin G [IgG], IgM, IgA, etc.) could be detected (1). It has been demonstrated previously that, at least in early responses, the antibodies to the nucleocapsid protein (N protein) predominate as assayed by Western blotting (3). Therefore, the N protein was chosen to be produced as a recombinant protein for establishing an antigen-capturing ELISA for SARS diagnosis.The SARS CoV N gene was obtained by reverse transcription PCR amplification from blood samples of a SARS patient in Beijing by using the following primer pair: 5Ј-CGCATATG TCTGATAATGGACCCCA-3Ј and 5Ј-CGGATCCTTATGC CTGAGTTGAATCAGCA-3Ј. The DNA fragment was then cloned into a T7 promoter-based prokaryotic expression vector, pET22b (Novagen). The resulting recombinant plasmid (pMG-N) was subjected to DNA sequencing and showed 100% identity with the N gene reported in the SARS CoV Toronto strain (GenBank accession number NC_004718). pMG-N was then transformed into Escherichia coli BL21a (DE3) and induced with 0.5 mmol of isopropyl-␤-D-thiogalactopyranoside (IPTG) (Sigma, St. Louis, Mo.) per liter for overexpression. The recombinant N prote...

Detection of Specific Antibodies to Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein for Serodiagnosis of SARS Coronavirus Pneumonia

“…Recently, Shi et al described the use of an antigen-capture ELISA for detection of SARSCoV nucleocapsid protein antibodies. With clinical criteria as the gold standard, this assay had a sensitivity of 89.6% for patients on days 11 to 61 of their illness (18). However, the authors did not show the sensitivities of the assay for IgG, IgM, and IgA detection.…”

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

“…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). Therefore, it is important to identify the immunoreactive epitope of the N protein or other proteins specific to the SARS-CoV for serodiagnosis of SARS.…”

“…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).…”

“…from humans and different animals can be detected simultaneously by an antigen-capturing ELISA (2,8), which makes it convenient to search for a natural animal reservoir for the SARS-CoV by use of the present N13-based antigen-capturing ELISA.…”

“…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).…”