Recombinant Protein-Based Assays for Detection of Antibodies to Severe Acute Respiratory Syndrome Coronavirus Spike and Nucleocapsid Proteins

Abstract

Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.The 2003 outbreak of severe acute respiratory syndrome (SARS) lasted fewer than 9 months, yet it had a major impact on public health and socioeconomics. Since the end of the SARS outbreak, there have been 17 identified SARS-associated coronavirus (SARS-CoV) infections, 6 from direct laboratory exposure, 1 of which resulted in community transmission to seven individuals, and 4 sporadic, community-acquired infections (9). Since additional cases may occur and could, if undetected, quickly lead to another global outbreak, it is important to continue to improve our ability to reliably monitor SARS-CoV infections (3,16,18).As with other coronaviruses, the spike (S) and nucleocapsid (N) proteins are abundantly expressed during virus infection and are most effective among the coronavirus structural proteins at inducing antibody responses (10,14,15,23). Previous studies have demonstrated the utility of anti-N and anti-S proteins in the diagnosis of SARS-CoV infections (2,5,12,21). In this study, we describe the evaluation and comparison of recombinant spike and nucleocapsid enzyme-linked immunosorbent assays (ELISAs) for specifically detecting SARS-CoV infection.The recombinant full-length SARS N gene was amplified from SARS-CoV RNA (Urbani strain), modified to contain a C-terminal His 6 tag, and cloned into the Venezuelan equine encephalitis virus replicon vector (17). Baby hamster kidney (BHK) cells were transfected with SARS N replicon RNA by electroporation. Cells were harvested, and expressed protein was purified by metal affinity chromatography and analyzed by silver staining and Western blot analysis for the appropriately sized (50-kDa) immunoreactive protein (8). The control antigen, the nontoxic 50-kDa C-terminal fragment of the botulinum neurotoxin serotype A (BoNt/HcA), was expressed as described above (7).The soluble codon-optimized SARS-CoV S glycoprotein (170 kDa; amino acids 1 to 1190; S1190) and the control antigen, truncated angiotensin-converting enzyme 2 (120 kDa; glycosylated; tACE-2), were cloned into pcDNA3.1 Myc/His and expressed in HEK-293T/17 cells. The proteins were purified using metal-affinity chromatography and analyzed as described by Babcock et al. (1).Recombinant SARS N and S protein indirect ELISAs were developed using a modified version of the inactivated wholevirus ELISA described by Ksiazek et al. (6). Briefly, ELISA plates (Immulon) were coated with either purified recombinant N protein (12.5 ng/well) and the control antigen BoNT/ HcA or purified His 6 /c-myc-tagged recombinant S1190 protein (12.5 ng/well) and the control antigen tACE-2. The plates wer...

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