摘要
新型冠状病毒感染人体后可引起发热、咳嗽等呼吸道症状,重症者可发生新型冠状病毒肺炎、严重急性呼吸综合征、甚至死亡。因此,快速、准确的识别新冠病毒的感染者,对于阻止病毒的持续传播非常重要。目前,新冠病毒抗原检测试剂产品大多以单个新冠病毒的N蛋白为目标抗原,存在一定的假阴性情况,本研究使用可识别多个新冠病毒结构蛋白抗原表位的多抗建立了一种双抗夹心ELISA方法,有望提高新冠病毒抗原检测的敏感性。利用课题组前期设计的一种新冠病毒抗原表位融合蛋白作为免疫原,搭配弗氏佐剂免疫新西兰大白兔,分析兔血清抗体的分泌情况及效价。利用制备的兔源多抗作为捕获抗体,以HRP标记的兔源多抗作为酶标抗体,建立一种用于检测新冠病毒抗原的双抗夹心ELISA检测方法。分别确定最佳捕获抗体、酶标抗体的稀释度以及最佳封闭液种类、封闭时间,初步评价方法的灵敏度和特异性,并比较不同样品储存条件对该ELISA方法检测效果的影响。新冠病毒抗原表位融合蛋白免疫兔后产生了高效价的特异性抗体,经纯化获得了相应的多抗。所建立的双抗夹心ELISA方法具有较好的检测限和线性范围,与常见的呼吸道病原体无交叉反应,有望用于新冠病毒的抗原检测。
SARS-CoV-2(severe acute respiratory syndrome coronavirus 2)can cause respiratory symptoms such as fever and cough when infected in humans.Severely ill people can develop COVID-19(Coronavirus Disease 2019),severe acute respiratory syn-drome,and even death.Therefore,rapid and accurate identification of those infected with SARS-CoV-2 and timely vaccination are very important to stop the continued spread of the virus.At present,most SARS-CoV-2 antigen detection reagents use a single SARS-CoV-2 N protein as the target antigen,which has certain false negative situations.In this study,a double antibody sandwich ELISA method was established by using multiple antibodies that can recognize multiple epitopes of SARS-CoV-2 structural proteins,which is expected to improve the sensitivity of SARS-CoV-2 antigen detection.A SARS-CoV-2 antigen epitope fusion protein designed by the authors′research group was used as the immunogen to immunize New Zealand white rabbits with Freund′s adjuvant,and the secretion and titer of rabbit serum antibodies were analyzed.Rabbit serum was purified by Protein A column to prepare polyclonal antibody and a double-antibody sandwich ELISA method for detecting SARS-CoV-2 antigen was established by using the prepared rabbit polyclonal antibody as the capture antibody and the rabbit polyclonal antibody labeled by HRP as the enzyme-labeled antibody.The dilution of the best captured antibody and enzymo-labeled antibody as well as the type and time of the best blocking solution were determined respectively.The sensitivity and specificity of the established ELISA method were preliminatively evaluated,and the influence of different sample storage conditions on the detection effect of the ELISA method was compared.After immunizing rabbits with the SARS-CoV-2 epitope fusion protein,specific antibodies with high potency were produced,and polyclonal antibodies corresponding to the SARS-CoV-2 epitope fusion protein were purified.The established double-antibody sandwich ELISA method has a good detection limit and linear range,and has no cross-reaction with common respiratory pathogens.The double-antibody sandwich ELISA method established in this study is expected to be used for antigen detection of SARS-CoV-2.
作者
庄素素
李巍
沈万鹏
李佳萌
刘文婧
徐应佳
赵楠
李越希
ZHUANG Su-su;LI Wei;SHEN Wan-peng;LI Jia-meng;LIU Wen-jing;XU Ying-jia;ZHAO Nan;LI Yue-xi(Nanjing Municipal Center for Disease Control and Prevention,Nanjing 210003,China;China Pharmaceutical University,Nanjing 211198,China;Huadong Research Institute for Medicine and Biotechniques,Nanjing 210002,China;School of Public Health,Nanjing Medical University,Nanjing 211166,China)
出处
《药物生物技术》
CAS
2023年第3期221-228,共8页
Pharmaceutical Biotechnology
基金
江苏省社会发展课题(No.BE2020631)。
