title: Evaluation of a safe and sensitive Spike protein-based immunofluorescence assay for the detection of antibody responses to SARS-CoV.
abstract: Previously, we have identified a truncated antigenic fragment named protein C [441 to 700 amino acids (a.a.)] as the immunodominant fragment of Spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). We have now successfully expressed protein C using the baculovirus system in S. frugiperda (Sf-9) cells. This recombinant baculovirus expressing protein C was first characterized using five SARS convalescent human sera and five normal human sera. The results showed that protein C is an authentic antigen against SARS-CoV antibody. Our Spike protein-based immunoflourescence assay (IFA) based on this recombinant baculovirus-Sf-9 system was further assessed with a panel of 163 clinical samples collected during the SARS epidemic in Singapore, which include samples from 21 clinically confirmed SARS, 42 non-SARS patient sera, and 100 normal sera. The results were compared to a commercial SARS IFA kit (EUROIMMUN, Germany) and a conventional IFA test performed in Singapore General Hospital. All of the 21 SARS-positive serum samples could be recognized by our IFA, giving a specificity and sensitivity of 100%, which was compatible with both whole virus-based IFA assays. No cross-reactivity with serum samples against infectious bronchitis virus (IBV) and transmissible gastroenteritis virus (TGEV) were detected in our assays. Thus, our Spike protein-based IFA could offer a safer procedure which can be performed in a BSL-2 laboratory as it could mimic the whole virus based-IFA without any loss of sensitivity and specificity. It is also more user-friendly and cost-effective than the whole virus-based IFA.
authors.affiliation: Animal Health Biotechnology, Temasek Life Sciences Laboratory, 1 Research link, National University of Singapore, 117604 Singapore.
authors.forename: Ivanus
authors.initials: I
authors.lastname: Manopo
authors.forename: Liqun
authors.initials: L
authors.lastname: Lu
authors.forename: Qigai
authors.initials: Q
authors.lastname: He
authors.forename: Li Lian
authors.initials: LL
authors.lastname: Chee
authors.forename: Shzu-Wei
authors.initials: SW
authors.lastname: Chan
authors.forename: Jimmy
authors.initials: J
authors.lastname: Kwang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood* / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics; D003429:Cross Reactions / Q000276:immunology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008562:Membrane Glycoproteins / Q000276:immunology; D008856:Microscopy, Fluorescence; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis*; D064370:Spike Glycoprotein, Coronavirus; D018411:Spodoptera / Q000166:cytology / Q000235:genetics; D014759:Viral Envelope Proteins / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus