Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome
Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome
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DOI：
10.1128/jcm.42.6.2629-2635.2004
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发表时间：
2004-06-01
影响因子：
9.4
通讯作者：
Yuen, KY
中科院分区：
文献类型：
--
作者：
Che, XY;Qiu, LW;Yuen, KY
诊断严重急性呼吸综合征（SARS）的快速抗原检测对于控制该病的管理至关重要。SARS相关冠状病毒（SARS-CoV）的核衣壳（N）蛋白在感染细胞培养滤液中大量表达，这一点可通过使用SARS患者恢复期血清的Western印迹法证实。本研究利用抗SARS冠状病毒N蛋白的单克隆抗体建立了灵敏的抗原捕获夹心酶联免疫吸附试验（ELISA），用于检测SARS冠状病毒。该方法采用了三种单克隆抗体的混合物捕获和兔多克隆抗体检测血清抗原在32例临床可能的SARS定义由世界卫生组织在中国广州流行期间。重组N蛋白用作标准品以建立约50 pg/ml的检测灵敏度。临床标本检测线性范围为100 ~ 3.2ng/ml。使用在不同时间点收集的一组血清，发现循环N抗原的量在症状发作后6至10天达到峰值。在13例血清学确诊的SARS患者中，在症状发作后的前10天内采集血液（11/13），检测的灵敏度为84.6%。在1，272名健康个体（1，253/1，272）中，该检测的特异性为98.5%。本检测方法与其他人和动物冠状病毒无交叉反应。本研究建立了一种灵敏、定量的抗原捕获ELISA方法，可用于SARS冠状病毒感染的早期诊断和病情监测。
A rapid antigen test for the diagnosis of severe acute respiratory syndrome (SARS) is essential for control of this disease at the point of management. The nucleocapsid (N) protein of SARS-associated coronavirus (SARS-CoV) is abundantly expressed in infected-cell culture filtrate as demonstrable by Western blotting using convalescent-phase sera from patients with SARS. We used monoclonal antibodies specifically directed against N protein to establish a sensitive antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV. The assay employed a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen in 32 cases of clinically probable SARS as defined by the World Health Organization during the epidemic in Guangzhou, China. Recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml. The linear range of detection in clinical specimens was from 100 pg/ml to 3.2 ng/ml. Using a panel of sera collected at different points in time, the amount of circulating N antigen was found to peak 6 to 10 days after the onset of symptoms. The sensitivity of the assay was 84.6% in 13 serologically confirmed SARS patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). The specificity of the assay was 98.5% in 1,272 healthy individuals (1,253 of 1,272). There was no cross-reaction with other human and animal coronaviruses in this assay. In conclusion, a sensitive and quantitative antigen capture ELISA was established for the early diagnosis and disease monitoring of SARS-CoV infection.