RESUMO
Severe acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. To study the humoral responses against SARS-CoV, we evaluated nucleocapsid (N) and spike (S) proteins-specific antibodies in patients' sera by Western blotting and enzyme-linked immunosorbent assay (ELISA). Recombinant N and S proteins of SARS-CoV were purified from transformed E. coli. Serum specimens from 40 SARS-CoV-infected patients in the convalescent phase were analyzed by Western blotting using the purified antigens. Serial serum specimens from 12 RT-PCR-confirmed SARS patients were assayed by ELISA using the recombinant N protein as coated antigen. By Western blotting, 97.5% of the SARS patients were positive for N protein-specific antibodies whereas only 47.5% of the samples were positive for S protein-specific antibodies. Using N protein-based ELISA, 10 out of the 12 patients were positive for N protein-specific antibodies and 6 of them showed seroconversion at mean of 16 days after onset of fever. Immunoblotting was useful for detecting the humoral immune response after SARS-CoV infection. Antibodies against SARS-CoV N protein appear at the early stage of infection, therefore, N protein-based ELISA could serve as a simple, sensitive, and specific test for diagnosing SARS-CoV infection.
Assuntos
Anticorpos Antivirais/sangue , Western Blotting , Ensaio de Imunoadsorção Enzimática , Glicoproteínas de Membrana/imunologia , Proteínas do Nucleocapsídeo/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Antivirais/biossíntese , Formação de Anticorpos , Antígenos Virais/genética , Antígenos Virais/imunologia , Clonagem Molecular , Proteínas do Nucleocapsídeo de Coronavírus , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Síndrome Respiratória Aguda Grave/diagnóstico , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/genéticaRESUMO
Severe acute respiratory syndrome (SARS) has raised a global alert since March 2003. After its causative agent, SARS-associated coronavirus (SARS-CoV), was confirmed, laboratory methods, including virus isolation, reverse transcriptase-polymerase chain reaction (RT-PCR), and serologic methods, have been quickly developed. In this study, we evaluated four serologic tests ( neutralization test, enzyme-linked immunosorbent assay [ELISA], immunofluorescent assay [IFA], and immunochromatographic test [ICT]) for detecting antibodies to SARS-CoV in sera of 537 probable SARS case-patients with correlation to the RT-PCR. With the neutralization test as a reference method, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.2%, 98.7%, 98.7%, and 98.4% for ELISA; 99.1%, 87.8%, 88.1% and 99.1% for IFA; 33.6%, 98.2%, 95.7%, and 56.1% for ICT, respectively. We also compared the recombinant-based western blot with the whole virus-based IFA and ELISA; the data showed a high correlation between these methods, with an overall agreement of >90%. Our results provide a systematic analysis of serologic and molecular methods for evaluating SARS-CoV infection.