This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by He, Q. Articles by Kwang, J. Search for Related Content PubMed PubMed Citation Articles by He, Q. Articles by Kwang, J.

Development of a Western Blot Assay for Detection of Antibodies against Coronavirus Causing Severe Acute Respiratory Syndrome

Animal Health Biotechnology, Temasek Life Science Laboratory, National University of Singapore,¹ Department of Rheumatology, Allergy and Immunology, Tan Tock Seng Hospital,² Virology Section, Department of Pathology, Singapore General Hospital,³ Environment Health Institute, National Environment Agency, Singapore, Republic of Singapore⁴

Received 23 September 2003/ Returned for modification 4 November 2003/ Accepted 4 December 2003

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.

This article has been cited by other articles:

• He, Q., Manopo, I., Lu, L., Leung, B. P., Chng, H. H., Ling, A. E., Chee, L. L., Chan, S.-W., Ooi, E. E., Sin, Y. L., Ang, B., Kwang, J. (2005). Novel Immunofluorescence Assay Using Recombinant Nucleocapsid-Spike Fusion Protein as Antigen To Detect Antibodies against Severe Acute Respiratory Syndrome Coronavirus. CVI 12: 321-328 [Abstract] [Full Text]  
• Guan, M., Chen, H. Y., Tan, P. H., Shen, S., Goh, P.-Y., Tan, Y.-J., Pang, P. H., Lu, Y., Fong, P. Y., Chin, D. (2004). Use of Viral Lysate Antigen Combined with Recombinant Protein in Western Immunoblot Assay as Confirmatory Test for Serodiagnosis of Severe Acute Respiratory Syndrome. CVI 11: 1148-1153 [Abstract] [Full Text]