Novel immunofluorescence assay using recombinant nucleocapsid-spike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus
Novel immunofluorescence assay using recombinant nucleocapsid-spike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus
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DOI：
10.1128/cdli.12.2.321-328.2005
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发表时间：
2005-02-01
期刊：
影响因子：
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通讯作者：
Kwang, J
中科院分区：
文献类型：
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作者：
He, Q;Manopo, I;Kwang, J
严重急性呼吸系统综合症（SARS）是由一种名为SARS冠状病毒（SARS- cov）的新型高传染性病毒引起的。在目前可用于检测SARS冠状病毒的血清学检测方法中，基于全病毒的免疫荧光检测（IFA）被认为是最敏感的检测方法之一，并在2003年新加坡SARS流行期间成为“金标准”。然而，由于对生物安全3级实验室的要求和实验室感染的风险，需要在传统的IFA中操作活的sars冠状病毒限制了其广泛应用。在此之前，我们已经在SARS-CoV的两个主要结构蛋白N和S蛋白中鉴定了两个免疫优势表位，命名为N195和Sc （He Q., k.h. Chong, h.h. Chug， B. Leung， A. E. Ling， T. Wei， S. W. Chan， E. E. Ooi和J. Kwang, clinin）。成岩作用。实验室。Immunol。11:417 - 422, 2004;吕丽玲，马诺波，梁伯平，郑宏宏，凌爱恩，齐丽丽，黄恩慈，陈世文，邝璟，克林。微生物学杂志。42(2):1570-1576,2004。在本研究中，N195-Sc融合蛋白在携带杂交基因的重组杆状病毒感染的昆虫（Sf9）细胞中在多面蛋白启动子的控制下高表达。用23个SARS患者的血清样本、20个自身免疫性疾病患者的血清样本和43个健康献血者的血清样本对基于产生融合蛋白的Sf9细胞的IFA进行了标准化。检出率与商用SARS-CoV IFA试剂盒（EUROIMMUN, Gross Groenau，德国）和新加坡总医院进行的传统IFA获得的检出率相当。我们的数据显示，新开发的IFA可以在23份SARS-CoV阳性血清样本中的22份中检测到SARS-CoV，并且在自身免疫性疾病患者和健康人的血清中检测没有假阳性结果。其检出率与两种基于全病毒的IFAs相同。因此，基于N-S融合抗原的新型IFA可能是目前基于全病毒的IFA诊断SARS-CoV感染的一个有吸引力的替代方案。
Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a "gold standard" during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chug, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422,2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.