新型冠状病毒N蛋白单克隆抗体制备及双抗体夹心ELISA抗原检测方法的建立

Preparation of A Monoclonal Antibody Against the Nucleocapsid Protein (NP) of SARS-CoV-2 and Establishment of A Double-Antibody Sandwich ELISA for NP Detection

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,R^(2)>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。

Severe acute respiratory syndrome-coronavirus disease(SARS-CoV)-2 infection can lead to coronavirus disease 2019(COVID-19). The latter has spread globally since it appeared in China in December2019. COVID-19 poses a serious threat to human life/health. A rapid,reliable,and easy-to-implement diagnosis is critical to combat the pandemic. The nucleocapsid protein(NP)of SARS-CoV-2 is an ideal target for viral antigen-based detection. The goat polyclonal antibody against SARS-CoV-2 and mouse monoclonal antibody against the NP of SARS-CoV-2 were prepared to establish a double-antibody sandwich enzyme-linked immunosorbent assay(ELISA)for quantitative detection of the NP. The ELISA system was optimized,and the linear range,sensitivity,specificity,repeatability,and accuracy were tested. The linear range was 1000–7.8 ng/mL,with correlation coefficient(R^(2))>0.99,and the lower limit of detection was 3.9 ng/mL. There was no reaction with the spike protein of SARS-CoV-2,Vero-cell debris,or influenza viruses,which indicated the high specificity of our method,and the sensitivity was 96.875%. The variation in intra-and inter-assay repeatability was 2.1% – 11.7% and 4.3% – 11.8%,respectively,and the accuracy for detecting samples was94.3% – 104.8%. These results showed that our method had high specificity,sensitivity,stability and accuracy,and could be used for quantitative detection of the NP of SARS-CoV-2.