title: Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome.
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.
authors.affiliation: Animal Health Biotechnology, Temasek Life Science Laboratory, National University of Singapore, Republic of Singapore.
authors.forename: Qigai
authors.initials: Q
authors.lastname: He
authors.forename: Kooi Hoong
authors.initials: KH
authors.lastname: Chong
authors.forename: Hiok Hee
authors.initials: HH
authors.lastname: Chng
authors.forename: Bernard
authors.initials: B
authors.lastname: Leung
authors.forename: Ai Ee
authors.initials: AE
authors.lastname: Ling
authors.forename: Ting
authors.initials: T
authors.lastname: Wei
authors.forename: Shzu-Wei
authors.initials: SW
authors.lastname: Chan
authors.forename: Eng Eong
authors.initials: EE
authors.lastname: Ooi
authors.forename: Jimmy
authors.initials: J
authors.lastname: Kwang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D015153:Blotting, Western / Q000379:methods*; D002645:Chickens; D003001:Cloning, Molecular; D004285:Dogs; D000939:Epitopes; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis* / Q000276:immunology*; D013552:Swine
chemical_list: D000914:Antibodies, Viral; D000939:Epitopes; D011994:Recombinant Proteins