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Dispatch
Nucleocapsid Protein as Early
Diagnostic Marker for SARS
Xiao-Yan Che,*
Wei Hao,* Yadi Wang,* Biao Di,† Kai Yin,* Yin-Chao Xu,* Chang-Sen Feng,*
Zhuo-Yue Wan,‡ Vincent C.C. Cheng,§ and Kwok-Yung Yuen§
*First Military Medical University, Guangzhou, People's Republic of China;
†Center for Disease Control and Prevention of Guangzhou, Guangzhou, People's
Republic of China; ‡Center for Disease Control and Prevention of Guangdong
Province, Guangzhou, People's Republic of China; and §The University of
Hong Kong, Hong Kong Special Administrative Region, People's Republic
of China
Suggested
citation for this article
Serum samples from
317 patients with patients with severe acute respiratory syndrome (SARS)
were tested for the nucleocapsid (N) protein of SARS-associated coronavirus,
with sensitivities of 94% and 78% for the first 5 days and 6–10 days
after onset, respectively. The specificity was 99.9%. N protein can
be used as an early diagnostic maker for SARS.
Early laboratory diagnosis of the severe acute respiratory syndrome–associated
coronavirus (SARS-CoV) is one step in preventing recurrence of a global
outbreak. The availability of the complete genomic sequence of SARS-CoV
has facilitated the development of a variety of diagnostic tests for SARS
(1). Reverse transcription–polymerase chain reaction
(RT-PCR) has been used as a rapid diagnostic test in most of the research
centers during the last epidemic (2–6). However, early
diagnosis of SARS remains a problem for nonresearch laboratories with
little experience in molecular testing. We have developed an antigen-capture
enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies
against the nucleocapsid (N) protein of SARS-CoV (7),
a predominant antigen produced in the infected cell-culture filtrate.
High levels of circulating N protein can be detected in the serum samples
of patients with SARS. We attempt to demonstrate the temporal profile
of the N protein and antibodies in serum samples from a large cohort of
patients with SARS during the acute and convalescent phases of the disease.
Our findings suggest that detecting N protein in serum can be used as
an early diagnostic marker for SARS.
The Study
During the 2003 SARS epidemic in Guangzhou, 420 serum specimens were
collected from 317 patients 1–90 days after the onset of symptoms. The
condition of all patients was diagnosed according to the World Health
Organization criteria and confirmed by seroconversion or a fourfold increase
in antibody titer against SARS-CoV by means of immunofluorescent testing.
The N protein–capture ELISA was performed (7). Briefly,
100 µL of serum was added to the wells of a microtiter plate coated
with a mixture of three anti-N protein monoclonal antibodies, and the
plates were incubated at 37°C for 60 min. After the plates were washed,
100 µL of anti-N rabbit antiserum was added to the wells, and the
plates were incubated at 37°C for 60 min. The wells were washed again
and incubated for 1 h at 37°C with 100 µL of peroxidase-conjugated
goat anti-rabbit immunoglobulin G (IgG). After the plates were washed,
100 µL of tetramethylbenzidine solution was added to each well.
The experiments involving the use of serum samples from patients with
SARS were performed within the safety cabinet of a biosafety level 2 laboratory.
The results for the 420 serum specimens tested by the N protein–capture
ELISA are shown in Figure 1. The N protein could
be detected as early as day 1 and until day 18. In the 146 serum samples
positive for N protein, the optical density (OD) value was highly variable
from one sample to another on the same day. The sensitivity of detection
was 94% (80 of 85 patients) with blood samples taken during the first
5 days and 78% (47 of 60 patients) for samples taken 6–10 days after onset
of symptoms. The detection rate of N protein decreased to 27% on days
11–20 after onset of symptoms. Serum N protein was never detected beyond
day 21. Using the same panel of the patient serum samples, we measured
the N protein–specific IgG (SARS-CoV N-IgG) and SARS-CoV–specific IgG
(SARS-CoV IgG) in serum samples by indirect ELISA, which progressively
increased from day 7 onward (Figure 2). With the
appearance of antibodies, the N protein detection rate decreased from
day 10 after the onset of symptoms. However, from day 7 to day 18, a high
level of N protein was still detectable in the serum samples from 11 patients
with a mean of OD values of 1.65 when the SARS-CoV N-IgG had already increased
to a level with a mean OD value of 1.18.
Serum samples from patients collected 4 years previously were used as
negative controls. Patients thought to have cases of SARS on admission
and later found to be uninfected by serologic testing, and healthcare
workers who had close contact with SARS patients were tested for circulating
N protein. Of the serum samples from non-SARS patients collected 4 years
previously (n = 400), only one was weakly positive for N protein (OD =
0.288). We tested a total of 110 acute-phase serum samples from 105 patients,
initially considered to have suspected SARS and later proven to be negative
for SARS-CoV by serologic testing of convalescent-phase serum samples
taken >28 days after onset of symptoms, and 315 serum samples from
healthcare workers. All were negative for the N protein by capture ELISA.
This finding resulted in a test specificity of 99.9% (1 of 825).
Conclusions
Our results suggest that N protein in the serum samples of SARS patients
can be detected as early as day 1 after disease onset. Although the level
of circulating N protein was highly variable from one person to another
from day 1 to day 18, development of SARS-CoV N-IgG appears not to be
affected by N protein during the acute phase of the infection, 7 days
after the onset of symptoms. The positive detection rate of N protein
in serum samples within the first 10 days of infection is higher than
that detected by RT-PCR (8,9). Furthermore, the variation
in the reported sensitivity and specificity of RT-PCR may be related to
the lack of standardization of the assay and the specimen collection (6).
Therefore, this viral antigen-capture ELISA may have greater sensitivity,
specificity, and ease and reliability of in-use performance than the nucleic
acid amplification assay. Further comparative studies with nucleic acid
amplification tests should be undertaken at clinical laboratories serving
acute-care hospitals where rapid SARS diagnosis is vital.
Acknowledgments
We thank Rongchang
Chen and Yuanda Xu for providing the clinical data for analysis.
This work was supported
by the special programs of SARS from the Ministry of Science and Technology
of the People's Republic of China and the Research Project of Guangdong
Province for SARS Prevention and Treatment.
Dr. Che is an immunologist
and professor at First Military Medical University, Guangzhou, China.
Her scientific interests are antibody technology and biotechnology.
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Suggested citation
for this article:
Che X-Y, Hao W, Wang
Y, Di B, Yin K, Xu Y-C, et al. Nucleocapsid protein as early diagnostic
marker for SARS. Emerg Infect Dis. [serial on the Internet]. 2004 Nov
[date cited]. Available from http://www.cdc.gov/ncidod/EID/vol10no11/04-0516.htm
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