Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome.
Identifieur interne : 002F24 ( PubMed/Corpus ); précédent : 002F23; suivant : 002F25Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome.
Auteurs : Qigai He ; Kooi Hoong Chong ; Hiok Hee Chng ; Bernard Leung ; Ai Ee Ling ; Ting Wei ; Shzu-Wei Chan ; Eng Eong Ooi ; Jimmy KwangSource :
- Clinical and diagnostic laboratory immunology [ 1071-412X ] ; 2004.
English descriptors
- KwdEn :
- Animals, Antibodies, Viral (analysis), Blotting, Western (methods), Chickens, Cloning, Molecular, Dogs, Epitopes, Humans, Recombinant Proteins (genetics), Recombinant Proteins (immunology), SARS Virus (genetics), SARS Virus (immunology), SARS Virus (isolation & purification), Severe Acute Respiratory Syndrome (diagnosis), Severe Acute Respiratory Syndrome (immunology), Swine.
- MESH :
- chemical , analysis : Antibodies, Viral.
- chemical , genetics : Recombinant Proteins.
- chemical , immunology : Recombinant Proteins.
- diagnosis : Severe Acute Respiratory Syndrome.
- genetics : SARS Virus.
- immunology : SARS Virus, Severe Acute Respiratory Syndrome.
- isolation & purification : SARS Virus.
- methods : Blotting, Western.
- Animals, Chickens, Cloning, Molecular, Dogs, Epitopes, Humans, Swine.
Abstract
To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.
DOI: 10.1128/cdli.11.2.417-422.2004
PubMed: 15013997
Links to Exploration step
pubmed:15013997Le document en format XML
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Ee" last="Ling">Ai Ee Ling</name></author><author><name sortKey="Wei, Ting" sort="Wei, Ting" uniqKey="Wei T" first="Ting" last="Wei">Ting Wei</name></author><author><name sortKey="Chan, Shzu Wei" sort="Chan, Shzu Wei" uniqKey="Chan S" first="Shzu-Wei" last="Chan">Shzu-Wei Chan</name></author><author><name sortKey="Ooi, Eng Eong" sort="Ooi, Eng Eong" uniqKey="Ooi E" first="Eng Eong" last="Ooi">Eng Eong Ooi</name></author><author><name sortKey="Kwang, Jimmy" sort="Kwang, Jimmy" uniqKey="Kwang J" first="Jimmy" last="Kwang">Jimmy Kwang</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2004">2004</date><idno type="RBID">pubmed:15013997</idno><idno type="pmid">15013997</idno><idno type="doi">10.1128/cdli.11.2.417-422.2004</idno><idno type="wicri:Area/PubMed/Corpus">002F24</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002F24</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome.</title><author><name sortKey="He, Qigai" sort="He, Qigai" uniqKey="He Q" first="Qigai" last="He">Qigai He</name><affiliation><nlm:affiliation>Animal Health Biotechnology, Temasek Life Science Laboratory, National University of Singapore, Republic of Singapore.</nlm:affiliation></affiliation></author><author><name sortKey="Chong, Kooi Hoong" sort="Chong, Kooi Hoong" uniqKey="Chong K" first="Kooi Hoong" last="Chong">Kooi Hoong Chong</name></author><author><name sortKey="Chng, Hiok Hee" sort="Chng, Hiok Hee" uniqKey="Chng H" first="Hiok Hee" last="Chng">Hiok Hee Chng</name></author><author><name sortKey="Leung, Bernard" sort="Leung, Bernard" uniqKey="Leung B" first="Bernard" last="Leung">Bernard Leung</name></author><author><name sortKey="Ling, Ai Ee" sort="Ling, Ai Ee" uniqKey="Ling A" first="Ai Ee" last="Ling">Ai Ee Ling</name></author><author><name sortKey="Wei, Ting" sort="Wei, Ting" uniqKey="Wei T" first="Ting" last="Wei">Ting Wei</name></author><author><name sortKey="Chan, Shzu Wei" sort="Chan, Shzu Wei" uniqKey="Chan S" first="Shzu-Wei" last="Chan">Shzu-Wei Chan</name></author><author><name sortKey="Ooi, Eng Eong" sort="Ooi, Eng Eong" uniqKey="Ooi E" first="Eng Eong" last="Ooi">Eng Eong Ooi</name></author><author><name sortKey="Kwang, Jimmy" sort="Kwang, Jimmy" uniqKey="Kwang J" first="Jimmy" last="Kwang">Jimmy Kwang</name></author></analytic><series><title level="j">Clinical and diagnostic laboratory immunology</title><idno type="ISSN">1071-412X</idno><imprint><date when="2004" type="published">2004</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Antibodies, Viral (analysis)</term><term>Blotting, Western (methods)</term><term>Chickens</term><term>Cloning, Molecular</term><term>Dogs</term><term>Epitopes</term><term>Humans</term><term>Recombinant Proteins (genetics)</term><term>Recombinant Proteins (immunology)</term><term>SARS Virus (genetics)</term><term>SARS Virus (immunology)</term><term>SARS Virus (isolation & purification)</term><term>Severe Acute Respiratory Syndrome (diagnosis)</term><term>Severe Acute Respiratory Syndrome (immunology)</term><term>Swine</term></keywords><keywords scheme="MESH" type="chemical" qualifier="analysis" xml:lang="en"><term>Antibodies, Viral</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Recombinant Proteins</term></keywords><keywords scheme="MESH" qualifier="diagnosis" xml:lang="en"><term>Severe Acute Respiratory Syndrome</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="immunology" xml:lang="en"><term>SARS Virus</term><term>Severe Acute Respiratory Syndrome</term></keywords><keywords scheme="MESH" qualifier="isolation & purification" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Blotting, Western</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Chickens</term><term>Cloning, Molecular</term><term>Dogs</term><term>Epitopes</term><term>Humans</term><term>Swine</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">15013997</PMID><DateCompleted><Year>2004</Year><Month>10</Month><Day>18</Day></DateCompleted><DateRevised><Year>2019</Year><Month>05</Month><Day>09</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">1071-412X</ISSN><JournalIssue CitedMedium="Print"><Volume>11</Volume><Issue>2</Issue><PubDate><Year>2004</Year><Month>Mar</Month></PubDate></JournalIssue><Title>Clinical and diagnostic laboratory immunology</Title><ISOAbbreviation>Clin. Diagn. Lab. Immunol.</ISOAbbreviation></Journal><ArticleTitle>Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome.</ArticleTitle><Pagination><MedlinePgn>417-22</MedlinePgn></Pagination><Abstract><AbstractText>To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>He</LastName><ForeName>Qigai</ForeName><Initials>Q</Initials><AffiliationInfo><Affiliation>Animal Health Biotechnology, Temasek Life Science Laboratory, National University of Singapore, Republic of Singapore.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Chong</LastName><ForeName>Kooi Hoong</ForeName><Initials>KH</Initials></Author><Author ValidYN="Y"><LastName>Chng</LastName><ForeName>Hiok Hee</ForeName><Initials>HH</Initials></Author><Author ValidYN="Y"><LastName>Leung</LastName><ForeName>Bernard</ForeName><Initials>B</Initials></Author><Author ValidYN="Y"><LastName>Ling</LastName><ForeName>Ai Ee</ForeName><Initials>AE</Initials></Author><Author ValidYN="Y"><LastName>Wei</LastName><ForeName>Ting</ForeName><Initials>T</Initials></Author><Author ValidYN="Y"><LastName>Chan</LastName><ForeName>Shzu-Wei</ForeName><Initials>SW</Initials></Author><Author ValidYN="Y"><LastName>Ooi</LastName><ForeName>Eng Eong</ForeName><Initials>EE</Initials></Author><Author ValidYN="Y"><LastName>Kwang</LastName><ForeName>Jimmy</ForeName><Initials>J</Initials></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Clin Diagn Lab Immunol</MedlineTA><NlmUniqueID>9421292</NlmUniqueID><ISSNLinking>1071-412X</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D000914">Antibodies, Viral</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance 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