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Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome.
Clin Diagn Lab Immunol. 2004 Mar; 11(2):417-22.CD

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.

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Authors+Show Affiliations

He Q
Animal Health Biotechnology, Temasek Life Science Laboratory, National University of Singapore, Republic of Singapore.
Chong KH
No affiliation info available
Chng HH
No affiliation info available
Leung B
No affiliation info available
Ling AE
No affiliation info available
Wei T
No affiliation info available
Chan SW
No affiliation info available
Ooi EE
No affiliation info available
Kwang J
No affiliation info available

MeSH

AnimalsAntibodies, ViralBlotting, WesternChickensCloning, MolecularDogsEpitopesHumansRecombinant ProteinsSevere acute respiratory syndrome-related coronavirusSevere Acute Respiratory SyndromeSwine

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15013997

Citation

He, Qigai, et al. "Development of a Western Blot Assay for Detection of Antibodies Against Coronavirus Causing Severe Acute Respiratory Syndrome." Clinical and Diagnostic Laboratory Immunology, vol. 11, no. 2, 2004, pp. 417-22.
He Q, Chong KH, Chng HH, et al. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin Diagn Lab Immunol. 2004;11(2):417-22.
He, Q., Chong, K. H., Chng, H. H., Leung, B., Ling, A. E., Wei, T., Chan, S. W., Ooi, E. E., & Kwang, J. (2004). Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clinical and Diagnostic Laboratory Immunology, 11(2), 417-22.
He Q, et al. Development of a Western Blot Assay for Detection of Antibodies Against Coronavirus Causing Severe Acute Respiratory Syndrome. Clin Diagn Lab Immunol. 2004;11(2):417-22. PubMed PMID: 15013997.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. AU - He,Qigai, AU - Chong,Kooi Hoong, AU - Chng,Hiok Hee, AU - Leung,Bernard, AU - Ling,Ai Ee, AU - Wei,Ting, AU - Chan,Shzu-Wei, AU - Ooi,Eng Eong, AU - Kwang,Jimmy, PY - 2004/3/12/pubmed PY - 2004/10/19/medline PY - 2004/3/12/entrez SP - 417 EP - 22 JF - Clinical and diagnostic laboratory immunology JO - Clin Diagn Lab Immunol VL - 11 IS - 2 N2 - 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. SN - 1071-412X UR - https://www.unboundmedicine.com/medline/citation/15013997/Development_of_a_Western_blot_assay_for_detection_of_antibodies_against_coronavirus_causing_severe_acute_respiratory_syndrome_ DB - PRIME DP - Unbound Medicine ER -
Grapherence [↓30 ↑17]

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