Novel Immunofluorescence Assay Using Recombinant Nucleocapsid-Spike Fusion Protein as Antigen To Detect Antibodies against Severe Acute Respiratory Syndrome Coronavirus
Identifieur interne : 004739 ( Main/Curation ); précédent : 004738; suivant : 004740Novel 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.
Descripteurs français
- KwdFr :
- Animaux, Anticorps antiviraux (immunologie), Anticorps antiviraux (sang), Données de séquences moléculaires, Dosage fluoroimmunologique (), Glycoprotéine de spicule des coronavirus, Glycoprotéines membranaires (génétique), Glycoprotéines membranaires (immunologie), Glycoprotéines membranaires (métabolisme), Humains, Protéines de fusion recombinantes (génétique), Protéines de fusion recombinantes (immunologie), Protéines de fusion recombinantes (métabolisme), Protéines de l'enveloppe virale (génétique), Protéines de l'enveloppe virale (immunologie), Protéines de l'enveloppe virale (métabolisme), Protéines nucléocapside (génétique), Protéines nucléocapside (immunologie), Protéines nucléocapside (métabolisme), Syndrome respiratoire aigu sévère (diagnostic), Séquence nucléotidique, Virus du SRAS (immunologie), Virus du SRAS (isolement et purification).
- MESH :
- diagnostic : Syndrome respiratoire aigu sévère.
- génétique : Glycoprotéines membranaires, Protéines de fusion recombinantes, Protéines de l'enveloppe virale, Protéines nucléocapside.
- immunologie : Anticorps antiviraux, Glycoprotéines membranaires, Protéines de fusion recombinantes, Protéines de l'enveloppe virale, Protéines nucléocapside, Virus du SRAS.
- isolement et purification : Virus du SRAS.
- métabolisme : Glycoprotéines membranaires, Protéines de fusion recombinantes, Protéines de l'enveloppe virale, Protéines nucléocapside.
- sang : Anticorps antiviraux.
- Animaux, Données de séquences moléculaires, Dosage fluoroimmunologique, Glycoprotéine de spicule des coronavirus, Humains, Séquence nucléotidique.
English descriptors
- KwdEn :
- Animals, Antibodies, Viral (blood), Antibodies, Viral (immunology), Base Sequence, Fluoroimmunoassay (methods), Humans, Membrane Glycoproteins (genetics), Membrane Glycoproteins (immunology), Membrane Glycoproteins (metabolism), Molecular Sequence Data, Nucleocapsid Proteins (genetics), Nucleocapsid Proteins (immunology), Nucleocapsid Proteins (metabolism), Recombinant Fusion Proteins (genetics), Recombinant Fusion Proteins (immunology), Recombinant Fusion Proteins (metabolism), SARS Virus (immunology), SARS Virus (isolation & purification), Severe Acute Respiratory Syndrome (diagnosis), Spike Glycoprotein, Coronavirus, Viral Envelope Proteins (genetics), Viral Envelope Proteins (immunology), Viral Envelope Proteins (metabolism).
- MESH :
- chemical , blood : Antibodies, Viral.
- chemical , genetics : Membrane Glycoproteins, Nucleocapsid Proteins, Recombinant Fusion Proteins, Viral Envelope Proteins.
- chemical , immunology : Antibodies, Viral, Membrane Glycoproteins, Nucleocapsid Proteins, Recombinant Fusion Proteins, Viral Envelope Proteins.
- chemical , metabolism : Membrane Glycoproteins, Nucleocapsid Proteins, Recombinant Fusion Proteins, Viral Envelope Proteins.
- diagnosis : Severe Acute Respiratory Syndrome.
- immunology : SARS Virus.
- isolation & purification : SARS Virus.
- methods : Fluoroimmunoassay.
- Animals, Base Sequence, Humans, Molecular Sequence Data, Spike Glycoprotein, 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.
Url:
DOI: 10.1128/CDLI.12.2.321-328.2005
PubMed: 15699428
PubMed Central: 549298
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PMC:549298Le document en format XML
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<term>Glycoprotéines membranaires (métabolisme)</term>
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<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>
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