摘要
核酸检测是检测新型冠状病毒(SARS-CoV-2)的主要检测方法,但该方法实验步骤复杂,检测时间较长,实验条件要求高,故本文研发了两种便捷、高效的免疫检测方法,作为核酸检测的重要补充.本研究通过提取新型冠状病毒核衣壳蛋白(N蛋白)作为抗原,制备出高特异性单克隆抗体,建立了N蛋白间接竞争ELISA法和胶体金免疫层析试纸条两种SARS-CoV-2免疫检测方法.通过优化间接竞争ELISA法的N蛋白包被量、抗体稀释倍数和稀释液pH等条件,得到该方法的灵敏度IC_(50)为(22.16±1.77)μg/L,最低检测限IC_(15)为(0.31±0.75)μg/L.通过优化胶体金免疫层析试纸条法的N蛋白和羊抗鼠二抗的稀释倍数、硝酸纤维素(NC)膜种类等条件,得到该方法的可视检测限(vLOD)和临界值分别为62μg/L和500μg/L.两种方法皆可在10 min左右得出结果,可以作为早期筛查的重要手段.本研究建立的两种新型冠状病毒抗原快速检测方法在SARS-CoV-2的早期检测、预防和控制领域具有良好的应用前景.
Nucleic acid testing is the primary test for the novel coronavirus(SARS-CoV-2).However,due to its complex experimental steps,long detection time and high experimental requirements,we developed two immune detection methods with convenient and efficient as an important supplement to nucleic acid detection.In our study,novel coronavirus nucleocapsid protein(N protein)was extracted as antigen to prepare monoclonal antibodies with high specificity,and indirect competitive ELISA method and colloidal gold immunochromatographic strip method for N protein were established.The sensitivity IC_(50) and the minimum detection limit IC_(15) of the indirect competitive ELISA method were respectively(22.16±1.77)μg/L and(0.31±0.75)μg/L by optimizing the encapsulation amount of N protein,dilution ratio of antibody and pH of dilution solution.The visual detection limit(vLOD)and critical value of colloidal gold immunochromatographic test strip method were respectively 62μg/L and 500μg/L by optimizing the dilution ratio of N protein and sheep anti-rat secondary antibody,nitrocellulose(NC)membrane type and other conditions.Moreover,the results could be obtained in about 10 min,which can be used as an important means of early screening.Therefore,our two immunological methods for rapid detection of novel coronavirus antigens have important reference value for early detection,prevention and control of SARS-CoV-2.
作者
江思思
刘俊伟
陆旸
JIANG Sisi;LIU Junwei;LU Yang(College of Food Science and Engineering,Tianjin University of Science&Technology,Tianjin 300457,China;Tianjin International Joint Academy of Biomedicine,Tianjin 300457,China)
出处
《天津科技大学学报》
CAS
2022年第4期23-29,共7页
Journal of Tianjin University of Science & Technology
基金
天津科技大学新型冠状病毒防治科研攻关项目(2020STCV0026)。
关键词
新型冠状病毒
快速检测
酶联免疫
免疫层析
胶体金
novel coronavirus
rapid detection
enzyme-linked immunization
immunochromatography
colloidal gold