Detection of Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein in SARS Patients by Enzyme-Linked Immunosorbent Assay

Abstract

We report the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of severe acute respiratory syndrome (SARS) coronavirus (CoV) nucleocapsid protein. The assay was carried out with hyperimmune polyclonal nucleocapsid-specific antibodies from guinea pigs and rabbits immunized with recombinant His 6 -tagged SARS CoV nucleocapsid protein. The assay was used for the detection of SARS CoV nucleocapsid protein in nasopharyngeal aspirate, urine, and fecal samples collected from patients with confirmed SARS between days 2 and 33 after the onset of illness. The ELISA was capable of detecting this protein in SARS CoV cell culture lysates at 15 50% tissue culture infective doses/ml but did not produce positive signals when tested with cell culture lysates of human coronaviruses OC43 and 229E. When tested with 120 nasopharyngeal aspirate, 100 urine, and 100 fecal specimens from hospitalized patients without SARS, the assay was shown to have high specificities-96.7, 99, and 96%, respectively. In an evaluation of clinical specimens from SARS patients, 34 (52%) of 66 nasopharyngeal aspirate samples from 50 patients, 5 (5%) of 94 urine samples from 94 patients, and 36 (55%) of 65 fecal samples from 65 patients tested positive for SARS CoV nucleocapsid protein. Nucleocapsid protein could be detected from days 6 to 24 in nasopharyngeal aspirate specimens, from days 11 to 31 in urine specimens, and from days 8 to 32 in fecal specimens after the onset of illness. Moreover, the protein could be detected in 25 (83%) of 30 nasopharyngeal aspirate specimens obtained from days 11 to 15 and in all 7 fecal specimens obtained from days 21 to 32. Since the present ELISA is more convenient and economical than reverse transcription-PCR, it may serve as an alternative tool for the early diagnosis of SARS CoV infection in laboratories with limited resources and expertise and for mass screening for the reservoir of SARS CoV. Further studies on serial clinical specimens should reveal the duration of nucleocapsid protein shedding and may reveal a higher detection rate in SARS patients.The severe acute respiratory syndrome (SARS) epidemic in 2003 affected 30 countries on five continents, with more than 8,000 cases and 750 deaths. A novel virus, the SARS coronavirus (CoV), was found be the etiological agent, and its genome has been completely sequenced (3-6). Recently, Guan et al. reported the isolation of SARS CoV-like viruses from Himalayan palm civets found in a live-animal market in the Guangdong Province of China, a result which implied that animals could be the reservoir for the ancestor of SARS CoV (2).Early diagnosis of SARS CoV infection is of paramount importance for both clinical management of patients and implementation of infection control measures. As a history of contact with SARS patients is not always present and the clinical presentation of SARS is nonspecific, diagnosis relies largely on laboratory tests. At the moment, the working "gold standard " for the laboratory diagnosis of SARS CoV infection is ant...

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