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
李妮;宋绍辉;钏鸿云;马磊;朱文勇;高菁霞;廖国阳;
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
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
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