Evaluation of antibody responses against SARS coronaviral nucleocapsid or spike proteins by immunoblotting or ELISA

Abstract

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.

Vaccination of mice with recombinant baculovirus expressing spike or nucleocapsid protein of SARS-like coronavirus generates humoral and cellular immune responses

“…Anti-N antibodies were present at high levels in SARS patients and persisted for a long time. In agreement, vectors that expressed full-length or truncated N proteins were highly immunogenic Guan et al, 2004;Huang et al, 2004;Leung et al, 2004;Tan et al, 2004;Woo et al, 2004). Hence, the S and N proteins may be the most promising vaccine candidates against SARS-CoV.…”

Immunofluorescence Assay for Detection of the Nucleocapsid Antigen of the Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus in Cells Derived from Throat Wash Samples of Patients with SARS

“…Nowadays, various laboratory diagnostic modalities such as virus isolation, reverse transcriptase-PCR (RT-PCR), antigen detection, and serological tests have been developed for the diagnosis of SARS-CoV infection (11,27). Since antibody against SARSCoV was found to be detectable at least 10 to 28 days after the onset of illness, detection of viral components appears to be the best option for early diagnosis (5,12,22,27). Virus isolation is insensitive and time consuming, and it requires special expertise and a biosafety level 3 facility (3,6,13,19).…”

“…To develop an antigen detection assay for SARS-CoV, we used the previously described polyclonal serum from a rabbit immunized with the recombinant N protein of SARS-CoV as the first antibody in the indirect immunofluorescence assay (IFA) (5). As the reagent control, spot slides prepared from SARS-CoV-infected Vero E6 cells were incubated with the pre-or postimmune serum, followed by the second antibody, a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit gamma globulin (Pierce Biotechnology, IL).…”

Different types of electrospun nanofibers and their effect on microfluidic‐based immunoassay

“…Most of the high sensitive medical diagnosis systems are designed on the basis of the fact that antibody and antigen interact each other with high specificity. Enzyme‐linked immunosorbent assay (ELISA) using two conjugate antibodies to bind an antigen is the most familiar technique widely applied in medical diagnostics, environmental analyses, and biochemical studies . However, conventional ELISA, which typically carried out in 96‐well microtiter plates, suffer from lacks such as complicated procedures and vigorous washing steps, consumption of large quantities of expensive reagents, and very long assay times .…”