SARS-CoV-2 S蛋白单克隆抗体的制备及其双抗体夹心ELISA检测方法的建立  

Preparation of monoclonal antibody against severe acute respiratory syndrome coronavirus 2 spike protein and development of double antibody sandwich ELISA

作　　者：李妮 龙柏霖 朱文勇[1] 钏鸿云 宋绍辉[1] 刘婧[1] 吴雅楠[1] 廖国阳[1] LI Ni;LONG Bai-lin;ZHU Wen-yong;CHUAN Hong-yun;SONG Shao-hui;LIU Jing;WU Ya-nan;LIAO Guo-yang(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650118,Yunnan Province,China;不详)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650118 [2]沈阳药科大学药学院,辽宁沈阳110016

出　　处：《中国生物制品学杂志》2022年第3期327-333,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金项目(81860281);中国医学科学院医学与健康科技创新工程(2020-I2M-2-014)。

摘　　要：目的 制备严重急性呼吸系统综合征冠状病毒2型(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)棘突蛋白(spike protein,S)单克隆抗体(简称单抗),建立S蛋白抗原含量双抗体夹心ELISA检测方法,并进行验证。方法 通过杂交瘤细胞融合技术制备单克隆抗体,并对单抗进行鉴定。用该单抗作为包被抗体,HRP标记的兔抗S蛋白多克隆抗体作为酶标抗体,建立双抗体夹心ELISA方法,检测SARS-CoV-2 S蛋白抗原含量。对方法的包被抗体浓度(10、5、2.5、1.25μg/mL)、酶标抗体浓度(4、2、1μg/mL)以及封闭液种类(未封闭、PBS稀释的1%BSA、2%BSA、1%BSA+1%蔗糖、2%BSA+2%蔗糖)进行优化,并对方法的线性范围及灵敏度、特异性和准确性进行验证。用建立的方法对10份疫苗生产工艺不同阶段已知S蛋白抗原含量的样本进行检测,计算该方法与中国医学科学院北京协和医学院医学生物学研究所新冠疫苗定量方法检测结果的符合率。结果 筛选出18株S蛋白特异性单克隆抗体,7B10A2细胞株制备的腹水抗体纯化后浓度为4.487 mg/mL,纯度大于90%,可特异性结合新冠病毒S蛋白上S1亚基;抗体亚型为IgG2b型,抗体效价为1︰128 000,效应浓度为0.137μg/mL。建立的双抗体夹心ELISA方法最适包被抗体和酶标抗体浓度分别为5和4μg/m L,最适封闭液为2%BSA+2%蔗糖;在2.5~160 U范围内,相关系数(R2)大于0.99,检测灵敏度为1.25 U;该方法特异性良好,与核衣壳蛋白(nucleocapsid protein,N)、BSA、PBS及流感病毒等不发生反应;准确性验证的回收率在92.10%~111.58%之间。用建立的方法检测已知含量样本符合率在94.2%~109%之间。结论 制备了SARS-CoV-2 S蛋白特异性单抗,并建立了适用于SARS-CoV-2 S蛋白抗原含量检测的双抗体夹心ELISA方法,可用于疫苗产品及其工艺阶段样品和其他样本中S蛋白含量的检测。Objective To prepare the monoclonal antibody(McAb)against spike(S)protein of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),and develop and verify a double antibody sandwich ELISA method for determination of spike protein antigen content. Methods McAb was prepared by hybridoma cell technology and identified. A double antibody sandwich ELISA using the prepared McAb as coating antibody and HRP-labeled rabbit anti-spike protein polyclonal antibody as detection antibody,and used for determination of SARS-CoV-2 spike protein antigen content. The concentrations of coating(10,5,2. 5 and 1. 25 μg/mL)and enzyme-labeled antibodies(4,2 and 1 μg/mL)as well as kinds of bloc king reagents(non-blocked,1% BSA diluted with PBS,2% BSA diluted with PBS,1% BSA + 1%sucrose,2% BSA + 2% sucrose) were optimized. The developed method was verified for linear range,sensitivity,specificity and accuracy. The 10 samples with known S protein antigen contents at various stages of production process were determined by the developed method,of which the coincidence rate of result to that by a quantitative determination method developed by Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College was calculated. Results Eighteen hybridom cell strains secreting S protein-specific McAb were screened. The ascites antibody prepared with 7B10A2 cell line reached a concentration of 4. 487 mg/m L and a purity of more than 90%after purification,which showed specific binding to the S1 subunit of S protein. The McAb was of an antibody subtype of IgG2b,of which the titer and effect concentration were 1 ∶ 128 000 and 0. 137 μg/mL respectively. The optimal concentrations of coating and enzyme-labeled antibodies were 5 and μg/mL respectively,while the optimal blocking reagent was 2% BSA + 2% sucrose. The linear range of the developed method was 2. 5 ~ 160 U,with a correlation coefficient(R2)of more than 0. 99 and a sensitivity of 1. 25 U. The method showed high specificity,with no reactions with nucleocaps

关 键 词：严重急性呼吸系统综合征冠状病毒2型 单克隆抗体 双抗体夹心ELISA 

分 类 号：R373.1[医药卫生—病原生物学]