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...

Catalase is a major effector in the defense of aerobic cells against oxidative stress. Recent studies have shown that catalase activity is stimulated by the c-Abl and Arg tyrosine kinases. Little, however, is otherwise known about the mechanisms responsible for catalase regulation. The present work demonstrates that mouse cells deficient in both c-Abl and Arg exhibit increased catalase stability. The results also show that catalase is subject to ubiquitination and degradation by the 26S proteosome. Significantly, ubiquitination of catalase is dependent on c-Abl- and Arg-mediated phosphorylation of catalase on both Y231 and Y386. In concert with these results, human 293 cells expressing catalase mutated at Y231 and Y386 exhibit attenuated levels of reactive oxygen species when exposed to hydrogen peroxide. These findings indicate that, in addition to stimulating catalase activity, c-Abl and Arg promote catalase degradation in the oxidative stress response.

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 ...