title: Novel immunofluorescence assay using recombinant nucleocapsid-spike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus.
abstract: Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a "gold standard" during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chng, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422, 2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.
authors.affiliation: Animal Health Biotechnology, Temasek Life Science Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604.
authors.forename: Qigai
authors.initials: Q
authors.lastname: He
authors.forename: Ivanus
authors.initials: I
authors.lastname: Manopo
authors.forename: Liqun
authors.initials: L
authors.lastname: Lu
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Leung
authors.forename: Hiok Hee
authors.initials: HH
authors.lastname: Chng
authors.forename: Ai Ee
authors.initials: AE
authors.lastname: Ling
authors.forename: Li Lian
authors.initials: LL
authors.lastname: Chee
authors.forename: Shzu-Wei
authors.initials: SW
authors.lastname: Chan
authors.forename: Eng Eong
authors.initials: EE
authors.lastname: Ooi
authors.forename: Yeo Lee
authors.initials: YL
authors.lastname: Sin
authors.forename: Brenda
authors.initials: B
authors.lastname: Ang
authors.forename: Jimmy
authors.initials: J
authors.lastname: Kwang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood* / Q000276:immunology; D001483:Base Sequence; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology / Q000302:isolation & purification*; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D008562:Membrane Glycoproteins; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV