肠道病毒71型灭活疫苗(Vero细胞)抗原含量检测方法的建立及应用  

Establishment and validation of a method to determine the antigen content of enterovirus 71 inactivated vaccine(Vero cells)

作　　者：赵丽丽 谢学超 陈磊[1] 李国顺[1] 顾美荣[1] 张改梅[1] ZHAO Lili;XIE Xuechao;CHEN Lei;LI Guoshun;GU Meirong;ZHANG Gaimei(Center of Research and Development,Beijing Minhai Biotechnology Co.,Ltd.,Center of Research of Novel Combination Vaccine Engineering Technology in Beijing,Beijing 102600,China)

机构地区：[1]北京民海生物科技有限公司研发中心北京市新型联合疫苗工程技术研究中心,北京102600

出　　处：《微生物学免疫学进展》2023年第6期21-28,共8页Progress In Microbiology and Immunology

摘　　要：目的建立肠道病毒71型(enterovirus type 71,EV-A71)灭活疫苗(Vero细胞)抗原含量检测方法并对其进行方法学验证及初步应用。方法以抗EV-A71兔多克隆抗体为包被抗体,辣根过氧化物酶(horseradish peroxidase,HRP)标记的抗EV-A71鼠单克隆抗体为检测抗体,建立双抗体夹心ELISA并验证其线性范围、专属性、准确度、精密度及耐用性等。通过检测EV-A71疫苗原液及研发生产工艺各中间制品的EV-A71抗原含量评价该方法的适用性。结果包被抗体与酶标抗体最佳稀释度分别为1∶5000和1∶10000。抗原为5~80 U/mL时,线性良好;与同为肠道病毒的其他抗原不存在交叉反应,专属性良好;对不同浓度抗原进行6次测定,其回收率在90%~100%,准确度良好;不同实验员对不同浓度抗原样品检测3次,CV均<11%,精密度良好;针对不同影响因素设计耐用性试验,回收率均在80%~120%,耐用性良好。该方法检测EV-A71疫苗原液和制备过程中的中间制品均具有良好的线性和平行性,表明该方法具有良好的适用性。结论建立了EV-A71疫苗(Vero细胞)抗原含量检测方法并进行了初步应用,为EV-A71疫苗和HFMD多价疫苗的研发及质量控制提供了可靠的检测方法。Objective To establish a method for the determination of antigen of enterovirus 71(EV-A71)inactivated vaccine(Vero cells).Methods Rabbit polyclonal antibody anti-EV-A71 was used as the coated antibody,and mouse monoclonal antibody anti-EV-A71 labeled with horseradish peroxidase(HRP)was used as the detection antibody and used to establish a Double antibody sandwich ELISA.The linear range,specificity,accuracy,precision,and durability of the method were verified.EV-A71 antigen content of the EV-A71 vaccine bulk and the intermediate products of the production process prepared were detected to evaluate the applicability of the method.Results The optimal dilutions of coated antibody and enzymic antibody were 1∶5000 and 1∶10000,respectively,and the linearity was good when the antigen was 5-80 U/mL.There was no cross-reaction with other antigens of enterovirus,and the specificity was reasonable.Different antigen concentrations were determined 6 times,and the recovery values were 90%-100%,with reasonable accuracy.Different antigen concentrations were tested 3 times by different experimentalists.The results showed that the coefficient of variation was less than 11%,and the precision was good.Durability tests were designed for different influencing factors,and the recovery values were 80%-120%,showing good durability.This method has good linearity and parallelism in detecting EV-A71 vaccine bulk and intermediate products in the preparation process,indicating that the method had good applicability.Conclusion A method for the determination of antigen content of EV-A71 vaccine(Vero cells)was established and validated,it provided a reliable tool for development and quality control of EV-A71 vaccine and polyvalent HFMD vaccine.

关 键 词：肠道病毒71型 双抗体夹心 酶联免疫法 抗原含量 单克隆抗体 多克隆抗体 

分 类 号：R373.2[医药卫生—病原生物学]