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.

FIG. 1.

Purified and expressed fragments of the nucleocapsid gene. (a) Amplified PCR products of the nucleocapsid gene and the relative locations of the truncated fragments. The full length and the truncated fragments of the nucleocapsid were amplified by PCR. N210, bp 1 to 630; N195, bp 684 to 1269; N170, bp 414 to 924 bp; N71, bp 414 to 627; N80A, bp 684 to 924; N80B, bp 1029 to 1269. Samples were loaded in a 1% agarose gel. (b) Expression of GST-fused full-length and truncated fragments of nucleocapsid protein in E. coli. Samples were loaded in a sodium dodecyl sulfate-12% polyacrylamide gel. Arrows indicate the positions of the proteins.

FIG. 2.

IgG detection of 10 representative positive samples and 2 representative negative samples. The purified N195 protein was immunoblotted onto a nitrocellulose membrane. Inactivated human antisera were used as the primary antibody at a 1:100 dilution, followed by a peroxidase-conjugated IgG secondary antibody. DAB was used as the horseradish peroxidase substrate, and the membrane was developed for 30 s. The arrow indicates the location of the N195 protein.