Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome
Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome
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DOI：
10.1128/cdli.11.2.417-422.2004
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发表时间：
2004-03-01
期刊：
影响因子：
--
通讯作者：
Kwang, J
中科院分区：
文献类型：
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作者：
He, QG;Chong, KH;Kwang, J
为了确定一个主要的抗原决定簇，用于严重急性呼吸综合征（SARS）冠状病毒感染的快速血清学诊断试验的发展，并研究在SARS冠状病毒感染的免疫反应，在人类，我们克隆了全长和六个截短片段的核衣壳基因，表达他们，并纯化谷胱甘肽S-转移酶标记的重组蛋白。用33份SARS冠状病毒阳性血清和66份SARS冠状病毒阴性血清及其它动物冠状病毒抗体检测重组蛋白的反应性。从核衣壳蛋白（N195）的C端截短的195个氨基酸的片段被鉴定出具有强的检测SARS冠状病毒抗体的能力。N195蛋白与鸡、猪和犬冠状病毒抗体之间未发现交叉反应。用N195蛋白建立Western blot检测274例临床盲法标本中SARS冠状病毒抗体。本试验的特异性和敏感性分别为98.3%和90.9%。我们的蛋白质印迹法和免疫荧光法（IFA）之间的相关性进行了分析。免疫印迹法和间接免疫荧光法检测SARS冠状病毒阳性血清结果一致。因此，N195蛋白被鉴定为在Western印迹和用于检测SARS冠状病毒感染的其他可能的测定中用作抗原的合适蛋白。
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.