抗埃博拉病毒核蛋白抗体的制备与双抗夹心ELISA检测方法的建立

doi:10.13523/j.cb.20171007

中国生物工程杂志

2017, Vol. 37

Issue (10): 53-59 DOI: 10.13523/j.cb.20171007

技术与方法

抗埃博拉病毒核蛋白抗体的制备与双抗夹心ELISA检测方法的建立

刘俊伟¹, 常瑞恒¹, 回鹏^1,2, 董士尚^1,2, 王金凤^1,2, 孙波¹, 杨诚^1,2

1. 天津国际生物医药联合研究院天津 300457;
2. 南开大学药学院天津 300071

Preparation of Anti-ebola Nucleoprotein Antibodies and Establishment of Sandwich ELISA Assay

LIU Jun-wei¹, CHANG Rui-heng¹, HUI Peng^1,2, DONG Shi-shang^1,2, WANG Jin-feng^1,2, SUN Bo¹, YANG Cheng^1,2

1. Tianjin International Joint Academy of Biomedicine, Tianjin 300457, China;
2. School of Pharmacy Nankai University, Tianjin 300071, China

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摘要： 目的：制备抗埃博拉病毒核蛋白（EBOV NP）单克隆抗体和多克隆抗体，建立针对EBOV NP的ELISA检测方法。方法：以重组EBOV NP免疫动物并制备多克隆抗体和单克隆抗体。在此基础上，通过优化抗体浓度、包被液等条件建立检测EBOV NP的双抗夹心ELISA方法。结果：制备出了兔多克隆抗体，筛选出2株可分泌单克隆抗体的鼠源杂交瘤细胞株。Western blot实验结果表明兔多抗与鼠单抗的结合区域均为N端1~35氨基酸。通过优化，建立了针对EBOV NP的双抗夹心ELISA检测方法。其线性范围是31.2~1 000 ng/ml，最低检测限为2.6 ng/ml。结论：制备出了抗EBOV核蛋白的高特异性多克隆抗体和单克隆抗体，建立了定量检测EBOV核蛋白的方法。

关键词： 埃博拉病毒; 抗体; 双抗夹心ELISA法; 核蛋白

Abstract: Objective:To prepare the anti-Ebola nucleoprotein monoclonal and polyclonal antibodies, and establish a method for determining the EBOV nucleoprotein. Methods:The rabbits and mice were immunized with EBOV nucleoprotein for preparing polyclonal and monoclonal antibodies, respectively. After optimized the conditions, such as the concentration of antibodies, coating solution and so on, the sandwich ELISA was established. Results:The polyclonal antibodies and two hybridoma cell lines that could produce monoclonal antibodies were prepared. The results of Western blot showed that the binding region of both polyclonal and monoclonal antibodies were in N terminal 1~35 amino acid of the nucleoprotein. The sandwich ELISA for detecting EBOV nucleoprotein has been optimized. The linear range of detection was 31.2~1 000 ng/ml. And the limit of detection was 2.6 ng/ml. Conclusions:The high specific polyclonal and monoclonal antibodies of anti-EBOV nucleoprotein were prepared. The quantitative method to detecting EBOV nucleoprotein was established.

Key words: Nucleoprotein Sandwich ELISA Ebola virus Antibody

收稿日期: 2017-03-23 出版日期: 2017-10-25

ZTFLH:

Q819

基金资助: 天津市科技计划项目（13ZCZDSY04200，13ZCZDSY03800）资助项目

通讯作者: 刘俊伟,ljwmars@163.com E-mail: ljwmars@163.com

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引用本文:

刘俊伟, 常瑞恒, 回鹏, 董士尚, 王金凤, 孙波, 杨诚. 抗埃博拉病毒核蛋白抗体的制备与双抗夹心ELISA检测方法的建立[J]. 中国生物工程杂志, 2017, 37(10): 53-59.

LIU Jun-wei, CHANG Rui-heng, HUI Peng, DONG Shi-shang, WANG Jin-feng, SUN Bo, YANG Cheng. Preparation of Anti-ebola Nucleoprotein Antibodies and Establishment of Sandwich ELISA Assay. China Biotechnology, 2017, 37(10): 53-59.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171007 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I10/53

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