Novel Immunofluorescence Assay Using Recombinant Nucleocapsid-Spike Fusion Protein as Antigen To Detect Antibodies against Severe Acute Respiratory Syndrome Coronavirus
Identifieur interne : 001286 ( Pmc/Checkpoint ); précédent : 001285; suivant : 001287Novel Immunofluorescence Assay Using Recombinant Nucleocapsid-Spike Fusion Protein as Antigen To Detect Antibodies against Severe Acute Respiratory Syndrome Coronavirus
Auteurs : Qigai He ; Ivanus Manopo ; Liqun Lu ; Bernard P. Leung ; Hiok Hee Chng ; Ai Ee Ling ; Li Lian Chee ; Shzu-Wei Chan ; Eng Eong Ooi ; Yeo Lee Sin ; Brenda Ang ; Jimmy KwangSource :
- Clinical and Diagnostic Laboratory Immunology [ 1071-412X ] ; 2005.
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.
Url:
DOI: 10.1128/CDLI.12.2.321-328.2005
PubMed: 15699428
PubMed Central: 549298
Affiliations:
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Leung</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Chng, Hiok Hee" sort="Chng, Hiok Hee" uniqKey="Chng H" first="Hiok Hee" last="Chng">Hiok Hee Chng</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ling, Ai Ee" sort="Ling, Ai Ee" uniqKey="Ling A" first="Ai Ee" last="Ling">Ai Ee Ling</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Chee, Li Lian" sort="Chee, Li Lian" uniqKey="Chee L" first="Li Lian" last="Chee">Li Lian Chee</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Chan, Shzu Wei" sort="Chan, Shzu Wei" uniqKey="Chan S" first="Shzu-Wei" last="Chan">Shzu-Wei Chan</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ooi, Eng Eong" sort="Ooi, Eng Eong" uniqKey="Ooi E" first="Eng Eong" last="Ooi">Eng Eong Ooi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sin, Yeo Lee" sort="Sin, Yeo Lee" uniqKey="Sin Y" first="Yeo Lee" last="Sin">Yeo Lee Sin</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ang, Brenda" sort="Ang, Brenda" uniqKey="Ang B" first="Brenda" last="Ang">Brenda Ang</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Kwang, Jimmy" sort="Kwang, Jimmy" uniqKey="Kwang J" first="Jimmy" last="Kwang">Jimmy Kwang</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">Clinical and Diagnostic Laboratory Immunology</title><idno type="ISSN">1071-412X</idno><idno type="eISSN">1098-6588</idno><imprint><date when="2005">2005</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>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.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Clin Diagn Lab Immunol</journal-id><journal-id journal-id-type="publisher-id">cdli</journal-id><journal-title>Clinical and Diagnostic Laboratory Immunology</journal-title><issn pub-type="ppub">1071-412X</issn><issn pub-type="epub">1098-6588</issn><publisher><publisher-name>American Society for Microbiology</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">15699428</article-id><article-id pub-id-type="pmc">549298</article-id><article-id pub-id-type="publisher-id">0105-04</article-id><article-id pub-id-type="doi">10.1128/CDLI.12.2.321-328.2005</article-id><article-categories><subj-group subj-group-type="heading"><subject>Microbial Immunology</subject></subj-group></article-categories><title-group><article-title>Novel Immunofluorescence Assay Using Recombinant Nucleocapsid-Spike Fusion Protein as Antigen To Detect Antibodies against Severe Acute Respiratory Syndrome Coronavirus</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>He</surname><given-names>Qigai</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Manopo</surname><given-names>Ivanus</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lu</surname><given-names>Liqun</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Leung</surname><given-names>Bernard P.</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Chng</surname><given-names>Hiok Hee</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Ling</surname><given-names>Ai Ee</given-names></name><xref ref-type="aff" rid="aff1">3</xref></contrib><contrib contrib-type="author"><name><surname>Chee</surname><given-names>Li Lian</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chan</surname><given-names>Shzu-Wei</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ooi</surname><given-names>Eng Eong</given-names></name><xref ref-type="aff" rid="aff1">4</xref></contrib><contrib contrib-type="author"><name><surname>Sin</surname><given-names>Yeo Lee</given-names></name><xref ref-type="aff" rid="aff1">5</xref></contrib><contrib contrib-type="author"><name><surname>Ang</surname><given-names>Brenda</given-names></name><xref ref-type="aff" rid="aff1">5</xref></contrib><contrib contrib-type="author"><name><surname>Kwang</surname><given-names>Jimmy</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="aff1">Animal Health Biotechnology, Temasek Life Science Laboratory, National University of Singapore,<label>1</label> Department of Rheumatology, Allergy & Immunology,<label>2</label> Department of Infectious Disease, Tan Tock Seng Hospital,<label>5</label> Virology Section, Department of Pathology, Singapore General Hospital,<label>3</label> Environment Health Institute, National Environment Agency, Singapore<label>4</label></aff><author-notes><fn id="cor1"><label>*</label><p>Corresponding author. Mailing address: Animal Health Biotechnology, Temasek Life Science Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604. Phone: 65-68727473. Fax: 65-68727007. E-mail: <email>kwang@tll.org.sg</email>.</p></fn></author-notes><pub-date pub-type="ppub"><month>2</month><year>2005</year></pub-date><volume>12</volume><issue>2</issue><fpage>321</fpage><lpage>328</lpage><history><date date-type="received"><day>7</day><month>5</month><year>2004</year></date><date date-type="rev-recd"><day>9</day><month>8</month><year>2004</year></date><date date-type="accepted"><day>22</day><month>11</month><year>2004</year></date></history><copyright-statement>Copyright © 2005, American Society for Microbiology</copyright-statement><copyright-year>2005</copyright-year><abstract><p>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.</p></abstract></article-meta></front></pmc><affiliations><list></list><tree><noCountry><name sortKey="Ang, Brenda" sort="Ang, Brenda" uniqKey="Ang B" first="Brenda" last="Ang">Brenda Ang</name><name sortKey="Chan, Shzu Wei" sort="Chan, Shzu Wei" uniqKey="Chan S" first="Shzu-Wei" last="Chan">Shzu-Wei Chan</name><name sortKey="Chee, Li Lian" sort="Chee, Li Lian" uniqKey="Chee L" first="Li Lian" last="Chee">Li Lian Chee</name><name sortKey="Chng, Hiok Hee" sort="Chng, Hiok Hee" uniqKey="Chng H" first="Hiok Hee" last="Chng">Hiok Hee Chng</name><name sortKey="He, Qigai" sort="He, Qigai" uniqKey="He Q" first="Qigai" last="He">Qigai He</name><name sortKey="Kwang, Jimmy" sort="Kwang, Jimmy" uniqKey="Kwang J" first="Jimmy" last="Kwang">Jimmy Kwang</name><name sortKey="Leung, Bernard P" sort="Leung, Bernard P" uniqKey="Leung B" first="Bernard P." last="Leung">Bernard P. Leung</name><name sortKey="Ling, Ai Ee" sort="Ling, Ai Ee" uniqKey="Ling A" first="Ai Ee" last="Ling">Ai Ee Ling</name><name sortKey="Lu, Liqun" sort="Lu, Liqun" uniqKey="Lu L" first="Liqun" last="Lu">Liqun Lu</name><name sortKey="Manopo, Ivanus" sort="Manopo, Ivanus" uniqKey="Manopo I" first="Ivanus" last="Manopo">Ivanus Manopo</name><name sortKey="Ooi, Eng Eong" sort="Ooi, Eng Eong" uniqKey="Ooi E" first="Eng Eong" last="Ooi">Eng Eong Ooi</name><name sortKey="Sin, Yeo Lee" sort="Sin, Yeo Lee" uniqKey="Sin Y" first="Yeo Lee" last="Sin">Yeo Lee Sin</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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