新型冠状病毒S蛋白抗体制备及双抗体夹心ELISA抗原检测方法的建立

Preparation of Antibodies Against the Spike Glycoprotein of SARS-CoV-2 and Establishment of a Double Antibody Sandwich ELISA for Detection of the Spike Glycoprotein

由SARS-CoV-2(Severe acute respiratory syndrome coronavirus 2)引起的新型冠状病毒肺炎(Corona virus disease 2019,COVID-19)自2019年底暴发以来,已导致上亿人次感染和数百万人死亡,严重威胁着全人类的生命健康。为了建立一种能快速对新冠病毒疫苗中S蛋白抗原进行定量检测的方法,本研究通过免疫山羊制备多克隆抗体作为包被抗体,通过杂交瘤细胞技术制备了S蛋白特异性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,并验证其线性范围、敏感性、特异性、稳定性及符合率。结果显示,建立的双抗体夹心ELISA检测方法线性范围为1U~64U,相关系数R^(2)大于0.99;特异性良好,敏感性为92.1%;批内和批间变异系数分别为2.5%~11.7%和1.3%~14.8%,检测已知背景样本符合率为96.7%。结果表明,该方法特异性好、敏感性高、且稳定性和准确性高,可用于新冠疫苗中S蛋白抗原含量测定。

The coronavirus disease 2019(COVID-19)pandemic poses a serious threat to human life and health.To establish a method for quantitative detection of detection of the spike glycoprotein of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)in vaccines.Goat anti-SARS-CoV-2 polyclonal antibody and mouse anti-SARS-CoV-2 spike glycoprotein monoclonal antibody were prepared to establish a double antibody sandwich enzyme-linked immunosorbent assay(ELISA)for detection of the spike glycoprotein.The ELISA system was optimized and the linear range,sensitivity,specificity,repeatability and coincidence rate were tested.The linear range was 1-64 U and correlation coefficient(R^(2))>0.99.There was no reaction with the nucleocapsid protein,Vero-cell debris or influenza viruses,etc,indicating the high specificity of our method.The sensitivity was 92.1%and the variations in intra-and inter-assay repeatability were 2.5%-11.7%and 1.3%-14.8%,respectively.The samples showed a coincidence rate of 96.7%with known background.Our method had high specificity,sensitivity,stability and accuracy,and could be used for determination of spikeglycoprotein antigen content in vaccine.