sIPV疫苗D抗原含量双抗体夹心ELISA检测方法的建立及验证  

Development and verification of double antibody sandwich ELISA method for determination of D antigen content in s IPV vaccine

作　　者：徐康维[1] 朱文慧 宋彦丽[1] 英志芳[1] 王剑锋[1] 权娅茹[1] 李长贵[1] XU Kangwei;ZHU Wenhui;SONG Yanli;YING Zhifang;WANG Jianfeng;QUAN Yaru;LI Changgui(National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,Beijing 102629,China;不详)

机构地区：[1]中国食品药品检定研究院,国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室,国家药品监督管理局生物制品质量研究与评价重点实验室,北102629 [2]北京市药品检验研究院病毒疫苗室,北京102206

出　　处：《中国生物制品学杂志》2023年第10期1230-1234,1241,共6页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2018ZX09738-005;2018ZX09737-003);北京市自然科学基金(L192009)。

摘　　要：目的 建立牛多抗对兔多抗夹心ELISA方法 检测Sabin株脊灰病毒灭活疫苗(Sabin strain inactivated poliovirus Vaccine,sIPV)D抗原含量,并对建立的方法 进行验证。方法 分别采用Ⅰ、Ⅱ、Ⅲ型sIPV疫苗原液作为抗原制备兔多抗,并通过间接ELISA法检测其效价及特异性。以牛多抗为包被抗体、兔多抗为显示抗体建立检测D抗原含量的双抗体夹心ELISA方法,并对方法 的准确度、精密度及D抗原专属性进行验证。用建立的方法 检测国内5家企业sIPV疫苗样品。结果 制备获得型别特异性好、效价高的Ⅰ、Ⅱ、Ⅲ型兔多抗,并成功建立了双抗体夹心ELISA方法。采用四参数拟合,3型别标准曲线均具有良好的线性关系,R^(2)均>0.99。Ⅰ、Ⅱ、Ⅲ型各试验加标回收率均为80%~120%,各浓度平均回收率分别为98.11%、97.41%、98.66%;重复性与中间精密度CV均<10%;能够对D/C抗原进行区分。建立的方法 对5家企业生产的sIPV疫苗均能够进行D抗原定量检测。结论 成功建立了检测sIPV疫苗D抗原含量的双抗体夹心ELISA方法,该方法 准确度、精密度良好,具有一定的D抗原特异性,能够对不同厂家生产的疫苗进行检测。Objective To develop and verify a sandwich ELISA method with bovine polyclonal antibody against rabbit polyclonal antibody for the determination of D antigen content of Sabin strain inactivated poliovirus vaccine(sIPV).Methods The rabbit polyclonal antibodies were prepared with sIPV vaccine bulks of type Ⅰ,Ⅱ and Ⅲ as antigens and detected for the titer and specificity by indirect ELISA.A double antibody sandwich ELISA with bovine polyclonal antibody as coating antibody and rabbit polyclonal antibody as detection antibody was developed to determine D antigen content,and the accuracy,precision and specificity to D antigen of the method were verified.sIPV vaccine samples from five domestic enterprises were detected by the developed method.Results The rabbit polyclonal antibodies for type Ⅰ,Ⅱ and Ⅲ sIPV with good specificity and high titer were prepared,and the double antibody sandwich ELISA method was successfully developed.Using four-parameter fitting,all three standard curves showed good linear relationship,and R^(2) values were more than 0.99.The spike recoveries of type Ⅰ,Ⅱ and Ⅲ D antigens were all within 80%~120%,with average values of98.11%,97.41% and 98.66%,respectively.The CVs of repeatability and intermediate precision were all below 10%.The method also distinguished D antigens from C antigens.The developed method determined the D antigen contents of sIPV vaccines from five enterprises.Conclusion A sandwich ELISA method for determination of D antigen content in sIPV vaccine was successfully developed with satisfying accuracy,precision and certain D antigen specificity,which can be used to detect vaccines produced by different manufacturers.

关 键 词：Sabin株脊髓灰质炎灭活疫苗 D抗原 酶联免疫吸附试验 抗体 

分 类 号：R183.3[医药卫生—流行病学] R392-3[医药卫生—公共卫生与预防医学]