Profile Of Specific Antibodies To Individual Proteins Of SARS Coronavirus And Development Of Antigen-capturing ELISA For Antibody Detection

Severe acute respiratory syndrome (SARS) has been witnessed a serious threat to public health and the economy development on a global scale. A novel coronavirus, the SARS coronavirus (SARS-CoV), was identified as the causative agent of SARS. Understanding the profile of specific antibodies to individual proteins of this virus in humans is critical to the development of recombinant vaccine and diagnostic tools. In the present study, we studied the antibody responses to individual proteins of the SARS-CoV and their neutralization activities, and then developed and evaluated an antigen-capturing enzyme-linked immunosorbent assay ( ELISA) based on recombinant nucleocapsid (N) protein for detection of the SARS-CoV antibodies. 1. Antibody responses to individual proteins of the SARS-CoV and their neutralization activitiesThirteen recombinant proteins associated with 4 structural proteins (S, E, M andN) and 5 putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of the SARS-CoV were cloned and expressed in Escherichia coli. They were purified and then printed onto aldehyded glass slides to fabricate protein microarrays for screening and monitoring their specific IgG antibodies in SARS patient's sera. Antibodies to proteins S, 3a, N and 9b were detected in the sera from convalescent-phase of SARS patients, whereas those to proteins E, M, 3b, 6 and 7a undetected. In the detectable specific antibodies, anti-N and anti-S were dominant and could persist in the sera ofSARS patients until 30 weeks. At week 30, the average fluorescence intensity values were about half of those at week 4. In addition, among the rabbit antisera to recombinant proteins S3 (aa 241-591), N (full length), 3a (aa 125-274) and 9b (full length), only anti-S 3 serum showed significant neutralizing activity for preventing the SARS-CoV infection in Vero E6 cells, with a neutralizing antibody titer of 1:708. The results indicate that anti-N and anti-S antibodies are potential diagnostic markers for the SARS-CoV infection, bacterially expressed recombinant proteins S3 and N could be used as candidate diagnostic antigens, and S3 could be used as a potential candidate for developing a subunit vaccine; serological evidence implies that two novel proteins, 3a and 9b, may be present in the SARS-CoV.2. Detection of the SARS-CoV antibodies with antigen-capturing ELISABased on the antigenicity analysis of different regions of the SARS-CoV N protein, an antigen-capturing ELISA was developed by using recombinant N protein. A recombinant truncated N protein N13 (aa 221-422) was proved to be a more specific coated antigen than the full-length N protein, and the N13-based antigen-capturing ELISA is more sensitive and specific than the whole virus lysate-based indirect ELISA. This method was then applied to serum samples from probable SARS cases. Of the 280 sera tested, the positive rates of the SARS-CoV antibodies were more than 91% after 21 days of the illness onset, and then up to 100% after 35 days of the illness onset. The present antigen-capturing ELISA appears to be a safe, economical, sensitive and specific test for serodiagnosis of SARS and epidemiological study.