李妮;宋绍辉;钏鸿云;马磊;朱文勇;高菁霞;廖国阳;

• 1:中国医学科学院北京协和医学院医学生物学研究所

摘要(Abstract)：

由新型冠状病毒（SARS-CoV-2）感染引起的新型冠状病毒肺炎（COVID-19）自暴发以来，严重威胁着人类健康。建立一种快速、可靠、易操作的可用于SARS-CoV-2感染早期诊断的检测方法，对于疫情防控至关重要。SARS-CoV-2核衣壳蛋白（Nucleocapsid protein,NP）是抗原检测的理想靶点。为了建立一种可快速对SARS-CoV-2 NP进行定量检测的诊断方法，本研究通过免疫山羊制备羊抗SARS-CoV-2多克隆抗体并作为包被抗体，通过杂交瘤细胞技术制备鼠抗NP单克隆抗体并作为检测抗体，建立了双抗体夹心ELISA抗原检测方法。对反应条件进行优化，并验证其线性范围及检测下限、敏感性、特异性、稳定性及准确度。结果显示，建立的方法检测线性范围为1 000 ng/mL～7.8 ng/mL,R2>0.99，检测下限为3.9 ng/mL；敏感性为96.875%，特异性良好，与Vero细胞、SARSCoV-2刺突蛋白、流感病毒等不发生交叉反应；批内和批间变异系数分别为2.1%～11.7%和4.3%～11.8%；检测样品回收率在94.3%～104.8%之间。结果表明，该方法特异性和稳定性好，敏感性和准确度高，可用于SARS-CoV-2NP定量检测。

关键词(KeyWords)： SARS-CoV-2;多克隆抗体;单克隆抗体;ELISA;核衣壳蛋白

Abstract:

Keywords:

基金项目(Foundation): 中国医学科学院医学与健康科技创新工程(项目号：2020-I2M-2-014)，题目：呼吸道病毒通用疫苗研发~~

作者(Authors): 李妮;宋绍辉;钏鸿云;马磊;朱文勇;高菁霞;廖国阳;

DOI: 10.13242/j.cnki.bingduxuebao.004114

参考文献(References)：

• [1] Cutler D M,Summers L H. The COVID-19 pandemic and the$16 trillion virus[J]. JAMA,2020,324(15):1495-1496.
• [2] Andreano E, Nicastri E, Paciello I, Pileri P,Manganaro N, Piccini G, Manenti A, Pantano E,Kabanova A,Troisi M,Vacca F,Cardamone D,De Santi C,Torres J L,Ozorowski G,Benincasa L,Jang H,Di Genova C,Depau L,Brunetti J,Agrati C,Capobianchi M R,Castilletti C,Emiliozzi A,Fabbiani M,Montagnani F,Bracci L,Sautto G,Ross T M,Montomoli E, Temperton N, Ward A B, Sala C,Ippolito G, Rappuoli R. Extremely potent human monoclonal antibodies from COVID-19 convalescent patients[J]. Cell,2021,184(7):1821-1835.
• [3] Sharfstein J M,Becker S J,Mello M M. Diagnostic testing for the novel coronavirus[J]. JAMA,2020,323(15):1437-1438.
• [4] Younes N,Al-Sadeq D W,Al-Jighefee H,Younes S,Al-Jamal O,Daas H I,Yassine H M,Nasrallah G K.Challenges in laboratory diagnosis of the novel coronavirus SARS-CoV-2[J]. Viruses, 2020,12(6):582.
• [5] Liu R,Han H,Liu F,Lv Z,Wu K,Liu Y,Feng Y,Zhu C. Positive rate of RT-PCR detection of SARSCoV-2 infection in 4880 cases from one hospital in Wuhan,China,from Jan to Feb 2020[J]. Clin Chim Acta,2020,505:172-175.
• [6] Diao B,Wen K,Zhang J,Chen J,Han C,Chen Y,Wang S,Deng G,Zhou H,Wu Y. Accuracy of a nucleocapsid protein antigen rapid test in the diagnosis of SARS-CoV-2 infection[J]. Clin Microbiol Infect,2021,27(2):281-289.
• [7] Liu W,Liu L,Kou G,Zheng Y,Ding Y,Ni W,Wang Q,Tan L,Wu W,Tang S,Xiong Z,Zheng S.Evaluation of nucleocapsid and spike protein-based enzyme-linked immunosorbent assays for detecting antibodies against SARS-CoV-2[J/OL]. J Clin Microbiol,2020,58(6):e00461-20.
• [8] Sun B,Feng Y,Mo X,Zheng P,Wang Q,Li P,Peng P,Liu X,Chen Z,Huang H,Zhang F,Luo W,Niu X,Hu P,Wang L,Peng H,Huang Z,Feng L,Li F,Zhang F,Li F,Zhong N,Chen L. Kinetics of SARSCoV-2 specific IgM and IgG responses in COVID-19patients[J]. Emerg Microbes Infect,2020,9(1):940-948.
• [9] Pan Y,Li X,Yang G,Fan J,Tang Y,Zhao J,Long X,Guo S,Zhao Z,Liu Y,Hu H,Xue H,Li Y.Serological immunochromatographic approach in diagnosis with SARS-CoV-2 infected COVID-19patients[J/OL]. J Infect,2020,81(1):e28-e32.
• [10]Wolfel R,Corman V M,Guggemos W,Seilmaier M,Zange S, Müller M A, Niemeyer D, Jones T C,Vollmar P, Rothe C, Hoelscher M, Bleicker T,Brünink S,Schneider J,Ehmann R,Zwirglmaier K,Drosten C, Wendtner C. Virological assessment of hospitalized patients with COVID-2019[J]. Nature,2020,581(7809):465-469.
• [11]Di B,Hao W,Gao Y,Wang M,Wang Y D,Qiu L W,Wen K,Zhou D H,Wu X W,Lu E J,Liao Z Y,Mei Y B,Zheng B J,Che X Y. Monoclonal antibodybased antigen capture enzyme-linked immunosorbent assay reveals high sensitivity of the nucleocapsid protein in acute-phase sera of severe acute respiratory syndrome patients[J]. Clin Diagn Lab Immunol,2005,12(1):135-140.
• [12]Sharfstein J M,Becker S J,Mello M M. Diagnostic testing for the novel coronavirus[J]. JAMA,2020,323(15):1437-1438.
• [13]张雪冰，潘纹玉，陈丽丽.新型冠状病毒实验室检测方法研究进展[J/OL].病毒学报，2021,37(02):428-434.DOI:10.13242/j.cnki.bingduxuebao.003898.