新型冠状病毒核蛋白抗原含量ELISA检测方法的建立及验证  

Development and verification of an ELISA method for determination of nucleocapsid protein content of severe acute respiratory syndrome coronavirus-2

作　　者：王文辉 宫立孟 林凤杰 李伟 王凯文 杨东升 郭靖 孟胜利 王泽鋆 申硕 WANG Wen-hui;GONG Li-meng;LIN Feng-jie;LI Wei;WANG Kai-wen;YANG Dong-sheng;GUO Jing;MENG Sheng-li;WANG Ze-jun;SHEN Shuo(Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430207,Hubei Province,China;不详)

机构地区：[1]武汉生物制品研究所有限责任公司,湖北武汉430207 [2]国家联合疫苗工程技术研究中心,湖北武汉430207 [3]武汉药品医疗器械检验所,湖北武汉430207

出　　处：《中国生物制品学杂志》2022年第8期968-973,980,共7页Chinese Journal of Biologicals

基　　金：国家科技攻关计划(2020YFC0842100);湖北省重点研发计划(2020BCB041);湖北省科技重大专项(2021ACB005).

摘　　要：目的制备抗重症急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)多克隆抗体和抗SARS-CoV-2核衣壳蛋白(nucleocapsid protein,N蛋白)单克隆抗体,建立针对SARS-CoV-2 N蛋白抗原含量ELISA检测方法,并对该方法进行验证。方法将SARS-CoV-2灭活病毒纯化抗原免疫日本大耳白兔制备兔多克隆抗体;免疫BALB/c小鼠,利用细胞融合技术制备杂交瘤细胞株,经蚀斑减少中和试验(plaque reduction neutralization test,PRNT)和间接ELISA法筛选获得抗SARS-CoV-2 N蛋白单克隆抗体。采用过碘酸钠氧化法对单克隆抗体5E11进行标记,偶联HRP分子,用方阵滴定法对包被抗体(50~800 ng/孔)和酶标抗体(1∶500~1∶8000)的工作浓度进行优化,建立SARS-CoV-2 N蛋白抗原含量ELISA检测方法,确定方法线性范围和最低检测限,并对其准确度、精密度、耐用性、特异性进行验证。用建立的方法检测3批SARS-CoV-2灭活疫苗工艺过程阶段样品中N蛋白抗原含量。结果选择高效价的SARS-CoV-2兔抗血清纯化后的多克隆抗体作为包被抗体,特异性强的单克隆抗体5E11作为酶标抗体,包被抗体最佳工作浓度为100 ng/孔,酶标抗体最佳工作浓度为1∶1000,建立了针对SARS-CoV-2 N蛋白抗原含量ELISA检测方法。该方法在1.6~200 WU/mL范围内,抗原含量与对应的A450/A630呈良好的线性关系,R2>0.99,最低检出限为1.6 WU/mL;高、中、低(100、50、25 WU/mL)浓度SARS-CoV-2内部参考品检测均值回收率分别为104.33%、101.33%和98.67%,CV分别为7.06%、7.47%和12.39%;重复性验证CV为8.54%,中间精密度验证CV为8.37%;耐用性验证回收率在88%~112%之间;该方法可特异性识别SARS-CoV-2全病毒Virion、重组N蛋白,与其他抗原均无交叉反应。结论成功建立了SARS-CoV-2 N蛋白抗原含量ELISA检测方法,该方法具有良好的线性相关性,准确度、精密度、特异性良好,可用于SARS-CoV-2灭活疫苗中N蛋白抗原含量的检测�Objective To prepare the polyclonal antibody(PcAb)against severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)and the monoclonal antibody(McAb)against nucleocapsid protein(NP)of SARS-CoV-2,and develop and verify an ELISA method for determination of NP content of SARS-CoV-2.Methods Rabbit PcAb was prepared by immunizing Japanese rabbits with purified antigen of inactivated SARS-CoV-2.BALB/c mice were immunized with the purified antigen,based on which hybridoma cell strain was prepared by cell fusion technique,and the McAb against NP of SARS-CoV-2 was screened by plaque reduction neutralization test(PRNT)and indirect ELISA.McAb 5E11 was labeled by sodium periodate oxidation and conjugated to HRP molecules.An ELISA method for determination of NP antigen content of SARS-CoV-2 was developed by optimization of working concentrations of coating antibody(50~800 ng/well)and enzyme-labeled antibody(1∶500~1∶8000)by chessboard titration,determined for linear range and minimum detection limit,and verified for accuracy,precision,durability and specificity.The NP antigen contents in samples taken at various steps of purification process of three batches of inactivated SARS-CoV-2 vaccine were determined by the developed method.Results An ELISA method for determination of NP antigen content of SARS-CoV-2 was established by using high-titer rabbit PcAb against SARS-CoV-2 coating antibody and high specific mouse McAb 5E11 against the NP of SARS-CoV-2 as enzyme-labeled antibody,at optimal working concentrations of 100 ng/well and 1∶1000 respectively.The antigen content at a range of 1.6~200 WU/mL showed good linear relationship to the corresponding A450/A630,with a R2 value of more than 0.99,while the minimum detection limit was 1.6 WU/mL.The mean recovery rates of internal reference of SARS-CoV-2 at high(100 WU/mL),moderate(50 WU/mL)and low(25 WU/mL)concentrations were 104.33%,101.33%and 98.67%,of which the CVs were 7.06%,7.47%and 12.39%,respectively.The CVs in verification for reproducibility and intermediate precisio

关 键 词：重症急性呼吸综合征冠状病毒2 核蛋白 单克隆抗体 ELISA 抗原检测 

分 类 号：R373.1[医药卫生—病原生物学]