Studier, F. healthy negative controls and 20 SARS patients. Separate immunoreactivity assays with each p-Cresol recombinant polypeptide demonstrated that a combination of N and S protein fragments was more suitable than the individual peptides for developing a serological assay for SARS-CoV. A new coronavirus (CoV) (order enzyme mix, supplemented with 3 mM Mg2+. The reaction was initiated with 2 U of the SuperScript III RT/Platinum enzyme mix (Invitrogen). For expression in expression host Rosetta BL21(DE3)pLysS. TABLE 1. Oligonucleotide primers used for RT-PCR amplification of recombinant fragments of nucleocapsid and spike proteins from SARS-CoVfor 30 min at 4C, and the supernatant was purified by using a nickel-nitrilotriacetic acid-agarose matrix column according to the manufacturer’s instructions (Qiagen). Protein manipulations. The apparent molecular mass of each polypeptide was determined by SDS-polyacrylamide gel electrophoresis (PAGE). The concentration of proteins was determined by UV absorbance at 280 nm using molar extinction coefficients, calculated as described by Gill and von Hippel (5). Polyclonal antibody against SARS-CoV. The polyclonal antibody was prepared by immunizing a rabbit with an intramuscular injection of 200 g of protein (50 g of each recombinant polypeptide: N1, N2, N3, and S251-683) in 1 ml of an emulsion containing 50% Freund’s complete adjuvant. The same dose was repeated after 15 days, p-Cresol p-Cresol and the same dose prepared in Freunds incomplete adjuvant was injected at 15 days after the second dose. The serum used corresponded to a blood sample drawn 3 months after the third injection. IFA. The immunofluorescence assay (IFA) used SARS-CoV-infected Vero-E6 cells from a commercial SARS-CoV IFA kit (Euroimmun). Briefly, the rabbit polyclonal serum obtained was first purified by protein A affinity chromatography (HiTrap protein A HP; Amersham BioLabs) and then labeled with fluorescein isothiocyanate (Sigma). A 1:1,000 dilution of the fluorescein-labeled rabbit polyclonal antibody was incubated for 30 min with SARS-CoV-infected cells. Noninfected cells were used as a control. After a wash, the reaction was visualized by fluorescence microscopy. ELISA measurement. Microtiter plates were coated with a mixture of the four recombinant polypeptides, diluted in phosphate-buffered saline (PBS) at a concentration of 1 1 to 5 g/ml each (3 g/ml N1, 2 g/ml N2, 1 g/ml N3, and 5 g/ml S251-683), and were incubated overnight at room temperature. Plates were blocked with newborn calf serum, washed, and then incubated with sera from SARS patients or healthy controls (both HAS3 at 1:100) in PBS containing p-Cresol newborn calf serum for 45 min at 37C. After a wash, a 1:100,000 dilution of peroxidase-conjugated goat anti-human immunoglobulin G (IgG) plus IgM (Jackson) was added and incubated at 37C for 30 min. Finally, the peroxidase reaction was visualized by using a tetramethylbenzidine-hydrogen peroxide solution as a substrate (Neogen Corporation). ELISAs with the single recombinant proteins, each applied to a plate, were also performed on six SARS serum samples. The same antigen dilutions were used, and the protocol described above was followed. Western blotting. Briefly, 0.25 g of each purified polypeptide was loaded onto an SDS-PAGE (12%) gel and transferred to a 0.2-m-pore-size nitrocellulose membrane using a Bio-Rad Mini Trans-Blot transfer unit. The membrane was first blocked with PBS containing 0.1% Tween 20 and 5% nonfat dry milk at room temperature for 2 h and then incubated with the specific primary antibody diluted 500-fold with washing buffer (PBS with 0.1% Tween 20). The membrane was washed five times and further incubated with horseradish peroxidase-labeled anti-rabbit IgG (Sigma). The proteins were visualized by a peroxidase reaction using a tetramethylbenzidine-hydrogen peroxide solution as the substrate. Calculations. The sensitivity of the assays was calculated as (number of samples with true-positive results)/(number of samples with true-positive results + number of samples with false-negative results). The specificity of the assays was calculated as (number of samples with true-negative results)/(number of samples with true-negative results + number of samples with false-positive results). Statistical analysis. MedCalc software (version 9.5.2.0; MedCalc Software, Mariakerke, Belgium) was used to analyze the ELISA results, and data were plotted in an interactive dot diagram. RESULTS RT-PCR amplification and expression and purification of the recombinant proteins. The cDNA synthesized by RT-PCR was cloned into the expression vectors. Four different polypeptides were prepared; three corresponded to the nucleocapsid protein of the virus, and the fourth corresponded to the spike protein. The amplified fragments were first cloned into pRSET,.