A pair of SARS-CoV-2 nucleocapsid protein monoclonal antibodies shows high specificity and sensitivity for diagnosis

An-Kai Zhu; Sa-Sa Li; Shu-Pei Yu; Zhao-Yong Zhang; Huan Li; Jing-Feng Li; Shan-Shan Gao; Heng Chen; Jincun Zhao; Run Shi; Mingjian Lu; Chaoyang Li

doi:10.1016/j.virs.2022.10.003

December 2022

. doi: 10.1016/j.virs.2022.10.003

Citation: An-Kai Zhu, Sa-Sa Li, Shu-Pei Yu, Zhao-Yong Zhang, Huan Li, Jing-Feng Li, Shan-Shan Gao, Heng Chen, Jincun Zhao, Run Shi, Mingjian Lu, Chaoyang Li. A pair of SARS-CoV-2 nucleocapsid protein monoclonal antibodies shows high specificity and sensitivity for diagnosis .VIROLOGICA SINICA, 2022, 37(6) : 942-945. http://dx.doi.org/10.1016/j.virs.2022.10.003

一对新型冠状病毒-2核壳蛋白检测具有高敏感度和特异性的单克隆抗体

祝岸凯 ^a ,
李洒洒 ^a ,
余书培 ^a ,
张昭勇 ^b ,
李欢 ^a ,
李景峰 ^a ,
高山山 ^a ,
陈恒 ^a ,
赵金存 ^b ,
师润 ^a,, ,
卢鸣剑 ^c,, ,
李朝阳 ^a,,

a. 广州医科大学附属肿瘤医院, 呼吸疾病国家重点实验室;
b. 呼吸疾病国家重点实验室, 国家呼吸疾病临床研究中心, 广州呼吸健康研究所, 广州医科大学附属第一医院;
c. 广州医科大学附属肿瘤医院介入科

通讯作者： 师润, runshi@gzhmu.edu.cn; 卢鸣剑, sange1234@sina.com; 李朝阳, chaoyangli@gzhmu.edu.cn
收稿日期： 2022-02-14
录用日期： 2022-10-17

摘要

虽然新型冠状病毒-2具有突变快传染性强的特点，但是其核壳蛋白高度保守，是抗原检测的重要靶点。针对新型冠状病毒-2的这个特点，我们克隆和表达了其核壳蛋白并制备了相应的单克隆抗体。这些单克隆抗体分别识别新型冠状病毒-2核壳蛋白的氨基端（1D5和2G11）和羧基端（1C7，2E11，3C6，和4F10）并且在免疫印迹和免疫荧光反应中能特异性的识别抗原。更重要的是，我们发现2G11/Bio-1C7这对单抗组合在酶联免疫反应中检测新型冠状病毒-2核壳蛋白的最低浓度为15ng每孔，同时2G11和我们表达的抗原对于新冠患者血样测检测敏感度高达97.4%而特异性为100%。因此，我们制备新型冠状病毒-2核壳蛋白特异性的单克隆抗体并建立了基于新型冠状病毒-2核壳蛋白的检测方法。
- 新型冠状病毒-2
- , 核壳蛋白及抗核壳蛋白抗体临床诊断
- , 单克隆抗体

A pair of SARS-CoV-2 nucleocapsid protein monoclonal antibodies shows high specificity and sensitivity for diagnosis

An-Kai Zhu ^a ,
Sa-Sa Li ^a ,
Shu-Pei Yu ^a ,
Zhao-Yong Zhang ^b ,
Huan Li ^a ,
Jing-Feng Li ^a ,
Shan-Shan Gao ^a ,
Heng Chen ^a ,
Jincun Zhao ^b ,
Run Shi ^a,, ,
Mingjian Lu ^c,, ,
Chaoyang Li ^a,,

a Affiliated Cancer Hospital and Institute of Guangzhou Medical University, State Key Laboratory of Respiratory Disease, Key Laboratory for Cell Homeostasis and Cancer Research of Guangdong High Education Institute, Guangzhou, 510095, China;
b State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510182, China;
c Department of Interventional Radiology, Affiliated Cancer Hospital and Institute of Guangzhou Medical University, Guangzhou, 510095, China

Corresponding author: Run Shi, runshi@gzhmu.edu.cn Mingjian Lu, sange1234@sina.com Chaoyang Li, chaoyangli@gzhmu.edu.cn
Received Date: 14 February 2022
Accepted Date: 17 October 2022

Abstract

Highlights
1. Seven monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid protein are produced, which can be applied in ELISA, Western blotting, and immunofluorescence staining.
2. A pair of mAbs, 2G11/bio-1C7, can detect SARS-CoV-2 nucleocapsid protein as low as 15 pg/well in the double sandwich ELISA.
3. The mAb, 2G11, shows 97.4% sensitivity and 100% specificity for diagnosing the human blood samples.

References
1. Almazán, F., Galán, C., Enjuanes, L., 2004. The nucleoprotein is required for efficient coronavirus genome replication. J. Virol. 78, 12683–12688.
2. Bai, C., Zhong, Q., Gao, G.F., 2021. Overview of SARS-CoV-2 genome-encoded proteins. Sci. China Life Sci. 65, 1–15.
3. Bar-On, Y.M., Flamholz, A., Phillips, R., Milo, R., 2020. Sars-cov-2 (Covid-19) by the numbers. Elife 9, 697–698.
4. Brian, D.A., Baric, R.S., 2005. Coronavirus genome structure and replication. In: Current Topics in Microbiology and Immunology, pp. 1–30.
5. Burbelo, P.D., Riedo, F.X., Morishima, C., Rawlings, S., Smith, D., Das, S., Strich, J.R., Chertow, D.S., Davey, R.T., Cohen, J.I., 2020. Sensitivity in detection of antibodies to nucleocapsid and spike proteins of severe acute respiratory syndrome coronavirus 2 in patients with coronavirus disease 2019. J. Infect. Dis. 222, 206–213.
6. Cohen-Dvashi, H., Weinstein, J., Katz, M., Ashkenazy-Eilon, M., Mor, Y., Shimon, A., Achdout, H., Tamir, H., Israely, T., Strobelt, R., Shemesh, M., Stoler-Barak, L., Shulman, Z., Paran, N., Fleishman, S.J., Diskin, R., 2022. Anti-SARS-CoV-2 immunoadhesin remains effective against Omicron and other emerging variants of concern. iScience, 105193.
7. Fehr, A.R., Perlman, S., 2015. Coronaviruses: an overview of their replication and pathogenesis. In: Maier, H.J., Bickerton, E., Britton, P. (Eds.), Coronaviruses: Methods and Protocols, Methods in Molecular Biology. Springer New York, New York, NY, pp. 1–23.
8. Ju, B., Zhang, Q., Ge, J., Wang, R., Sun, J., Ge, X., Yu, Jiazhen, Shan, S., Zhou, B., Song, S., Tang, X., Yu, Jinfang, Lan, J., Yuan, J., Wang, H., Zhao, Juanjuan, Zhang, S., Wang, Y., Shi, X., Liu, L., Zhao, Jincun, Wang, X., Zhang, Z., Zhang, L., 2020. Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature 584, 115–119.
9. Kang, S., Yang, M., Hong, Z., Zhang, L., Huang, Z., Chen, X., He, S., Zhou, Ziliang, Zhou, Zhechong, Chen, Q., Yan, Y., Zhang, C., Shan, H., Chen, S., 2020. Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites. Acta Pharm. Sin. B 10, 1228–1238.
10. Ludwig, S., Zarbock, A., 2020. Coronaviruses and SARS-CoV-2: a brief overview. Anesth. Analg. 131, 93–96.
11. Naqvi, A.A.T., Fatima, K., Mohammad, T., Fatima, U., Singh, I.K., Singh, A., Atif, S.M., Hariprasad, G., Hasan, G.M., Hassan, M.I., 2020. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: structural genomics approach. Biochim. Biophys. Acta, Mol. Basis Dis. 1866, 165878.
12. Ni, L., Ye, F., Cheng, M.L., Feng, Y., Deng, Y.Q., Zhao, H., Wei, P., Ge, J., Gou, M., Li, X., Sun, L., Cao, T., Wang, P., Zhou, C., Zhang, R., Liang, P., Guo, H., Wang, X., Qin, C.F., Chen, F., Dong, C., 2020. Detection of SARS-CoV-2-specific humoral and cellular immunity in COVID-19 convalescent individuals. Immunity 52, 971–977.e3.
13. Peng, Y., Du, N., Lei, Y., Dorje, S., Qi, J., Luo, T., Gao, G.F., Song, H., 2020. Structures of the SARS -CoV-2 nucleocapsid and their perspectives for drug design. EMBO J. 39, 1–12.
14. Sun, X., Yi, C., Zhu, Y., Ding, L., Xia, S., Chen, X., Liu, M., Gu, C., Lu, X., Fu, Y., Chen, S., Zhang, T., Zhang, Y., Yang, Z., Ma, L., Gu, W., Hu, G., Du, S., Yan, R., Fu, W., Yuan, S., Qiu, C., Zhao, C., Zhang, X., He, Y., Qu, A., Zhou, X., Li, X., Wong, G., Deng, Q., Zhou, Q., Lu, H., Ling, Z., Ding, J., Lu, L., Xu, J., Xie, Y., Sun, B., 2022. Neutralization mechanism of a human antibody with pan-coronavirus reactivity including SARSCoV-2. Nat. Microbiol. 7, 1063–1074.
15. Wang, S., Wu, Y., Wang, Y., Chen, Z., Ying, D., Lin, X., Liu, C., Lin, M., Zhang, J., Zhu, Y., Guo, S., Shang, H., Chen, X., Qiang, H., Yin, Y., Tang, Z., Zheng, Z., Xia, N., 2022. Potential of antibody pair targeting conserved antigenic sites in diagnosis of SARSCoV-2 variants infection. J. Virol. Methods 309, 114597.
16. Xiang, F., Wang, X., He, X., Peng, Z., Yang, B., Zhang, J., Zhou, Q., Ye, H., Ma, Y., Li, H., Wei, X., Cai, P., Ma, W.L., 2020. Antibody detection and dynamic characteristics in patients with coronavirus disease 2019. Clin. Infect. Dis. 71, 1930–1934.
17. Xu, X., Chen, P., Wang, J., Feng, J., Zhou, H., Li, X., Zhong, W., Hao, P., 2020. Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission. Sci. China Life Sci. 63, 457–460.
18. Zeng, W., Liu, G., Ma, H., Zhao, D., Yang, Yunru, Liu, M., Mohammed, A., Zhao, C., Yang, Yun, Xie, J., Ding, C., Ma, X., Weng, J., Gao, Y., He, H., Jin, T., 2020. Biochemical characterization of SARS-CoV-2 nucleocapsid protein. Biochem. Biophys. Res. Commun. 527, 618–623.
19. Zhou, P., Yang, X. Lou, Wang, X.G., Hu, B., Zhang, L., Zhang, W., Si, H.R., Zhu, Y., Li, B., Huang, C.L., Chen, H.D., Chen, J., Luo, Y., Guo, H., Jiang, R., Di, Liu, M.Q., Chen, Y., Shen, X.R., Wang, X., Zheng, X.S., Zhao, K., Chen, Q.J., Deng, F., Liu, L.L., Yan, B., Zhan, F.X., Wang, Y.Y., Xiao, G.F., Shi, Z.L., 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273.
Proportional views
Electronic Supplementary Material

10.1016j.virs.2022.10.003-ESM.docx