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...

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