摘要
目的制备重组严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)蛋白疫苗抗原含量通用检测试剂盒,并进行验证。方法选用中国食品药品检定研究院(简称中检院)制备的羊抗S蛋白多克隆抗体作为包被抗体,从4株单克隆抗体(14C8、15F9、17A7和20D8)中筛选出1株具有受体结合域(receptor-binding domain,RBD)结合活性高,且广谱抗主要突变株的单克隆抗体作为酶标抗体(用HRP标记),制备重组SARS-CoV-2疫苗抗原含量的双抗体夹心ELISA法通用检测试剂盒,并采用棋盘滴定法对包被抗体稀释度(1∶125~1∶4000)和酶标抗体稀释度(1∶250~1∶32000)进行优化。验证试剂盒的专属性、线性范围、准确性、精密性及耐用性。将制备的通用检测试剂盒分发给12个实验室,检测各实验室自制的不同表达系统(CHO细胞、毕赤酵母、Sf9细胞或大肠埃希菌)和目的蛋白(RBD或S蛋白)的15批重组SARS-CoV-2蛋白疫苗原液(包括11批以WT株为参考序列设计的原液及4批以Beta、Gamma和Delta变异株设计的原液)。结果确定包被抗体最佳稀释度为1∶500,单克隆抗体20D8作为酶标抗体,最佳稀释度为1∶4000。通用检测试剂盒与严重急性呼吸综合征(SARS)和中东呼吸综合征(Middle East respiratory syndrome,MERS)病毒的重组S蛋白无交叉反应;第1代重组SARS-CoV-2蛋白疫苗抗原国家标准品(简称国家标准品)浓度在0.16~2.50 U/mL范围内,与A450/630呈良好的线性关系,线性方程为:y=0.791 x-0.1004,R2=0.9937;0.16~2.50 U/mL国家标准品重复6次检测结果回收率均在95%~104%之间,变异系数(coefficient of variation,CV)均<15%;2名实验员在不同时间重复3次检测结果的CV为4.4%~6.6%;在不同温度及时间条件下检测结果回收率均在80%~120%范围内。15批重组SARS-CoV-2蛋白疫苗原液抗原含量的检测结果与国家标准品均具有良好的平行性。结论本研究建立的抗原含量通用检测�
Objective To prepare and verify a uniform antigen content detection kit for recombinant protein vaccines against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods Using goat anti-S protein polyclonal antibody prepared by National Institutes for Food and Drug Control(NIFDC)as coating antibody,one of four monoclonal antibodies(14C8,15F9,17A7 and 20D8)with high receptor-binding domain(RBD)binding activity and broad-spectrum resistance against major variants as HRP-labeled antibody,a uniform double-antibody sandwich ELISA kit for antigen content detection of recombinant SARS-CoV-2 vaccine was prepared,and the dilution of coating antibody(1∶125~1∶4000)and enzymelabeled antibody(1∶250~1∶32000)were optimized by chessboard titration.The specificity,linear range,accuracy,precision and durability of the kit were verified.The prepared uniform detection kits were distributed to 12 laboratories to detect15 batches of recombinant SARS-CoV-2 protein vaccine stock solutions(including 11 batches of stock solutions designed with WT strain and 4 batches of designed with Beta,Gamma and Delta variants as reference sequence)from different expression systems(CHO cells,Pichia pastoris,Sf9 cells or E.coli)and target proteins(RBD or S protein)prepared by each laboratory.Results Monoclonal antibody 20D8 was used as the enzyme-labeled antibody with the optimal dilution of 1:4000,and the optimal dilution of coating antibody was 1:500.The uniform detection kit showed no cross reaction with recombinant S protein of severe acute respiratory syndrome(SARS)and Middle East respiratory syndrome(MERS).The first generation national standard for recombinant SARS-CoV-2 protein vaccine antigen(national standard for short)showed a concentration of 0.16~2.50 U/mL with a good linear relationship with A450/630,and the linear equation was:y=0.791 x-0.1004,R2=0.9937;The recoveries of 0.16~2.50 U/mL national standard in 6 repeated tests were 95%~104%and the coefficients of variation(CVs)were less than 15%;The CVs in 3 repeated tests b
作者
安超强
刘东
卞莲莲
刘明琛
么山山
徐康维
李长贵
吴星
毛群颖
高帆
徐苗
姜崴
梁争论
AN Chaoqiang;LIU Dong;BIAN Lianlian;LIU Mingchen;YAO Shanshan;XU Kangwei;LI Changgui;WU Xing;MAO Qunying;GAO Fan;XU Miao;JIANG Wei;LIANG Zhenglun(National Institutes for Food and Drug Control,Beijing 102629,China;不详)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2023年第4期411-418,共8页
Chinese Journal of Biologicals
基金
国家科技重大专项(2018ZX09101-001)
中国医学科学院医学与健康科技创新工程(2021-I2M-5-005)。
关键词
严重急性呼吸综合征冠状病毒2
重组蛋白疫苗
抗原含量
通用试剂盒
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)
Recombinant protein vaccines
Antigen content
Uniform kit
