摘要
目的建立定量检测肠道病毒71型(enterovirus 71,EV71)病毒样颗粒(virus-like particle,VLP)疫苗抗原含量的检测方法,并进行验证。方法用EV71 VLP抗原免疫日本大耳白兔,制备多克隆抗体,并进行Protein A亲和层析纯化。以纯化的抗EV71 VLP多克隆抗体为包被抗体,筛选最佳包被浓度及酶标抗体稀释倍数,建立定量检测EV71 VLP抗原含量的双抗体夹心ELISA法,并对该方法进行准确度、精密度验证,确定方法的线性范围和定量限。结果抗EV71多克隆抗体包被浓度5μg/mL,酶标抗体稀释度1∶10000条件下,建立了检测EV71 VLP抗原含量的双抗体夹心ELISA法。抗原标准品浓度在0.5~8 U/mL范围内,其对数值与对应的A450值对数值呈良好的线性关系,R^(2)=0.9927,定量限为2 ng/mL;准确度验证回收率为81%~106%;精密度验证相对标准偏差(RSD)为4.96%~12.04%。结论建立了用于EV71 VLP抗原定量检测的双抗体夹心ELISA法,有望用于EV71 VLP疫苗抗原含量的初步检测。
Objective To develop and validate a quantitative determination method for the antigen content of enterovirus 71(EV71)virus-like particle(VLP)vaccine.Methods Polyclonal antibodies were prepared by immunizing Japanese white rabbits with EV71 VLP antigen and purified by Protein A affinity chromatography.The purified anti-EV71 VLP polyclonal antibody was used as the coating antibody,of which the concentration for coating and dilution factor of enzymlabeled antibody were optimized.A double-antibody sandwich ELISA method for determination of EV71 VLP antigen content was developed,validated for accuracy and precision,and determined for linear range and quantitative limit.Results The optimal concentration of anti-EV71 VLP polyclonal antibody for coating was 5μg/mL,while the optimal dilution factor of enzyme-labeled antibody was 1∶10000,at which a double-antibody sandwich ELISA method for determination of EV71 VLP antigen content was developed.The log of concentration of standard antigen at a range of 0.5~8 U/mL showed a good linear relationship with that of the corresponding A450 value(R^(2)=0.9927),and the limit of quantification was 2 ng/mL.The recoveries in validation for accuracy was 81%~106%,while the relative standard deviation(RSD)in validation for precision was 4.96%~12.04%.Conclusion A double-antibody sandwich ELISA method was developed for the quantitative determination of EV71 VLP antigen,which was expected to be used for the preliminary determination of EV71 VLP vaccine antigen.
作者
施歌
石亮
王翠艳
白鹭
褚彦飞
史赫
谷铁军
刘大维
包小华
SHI Ge;SHI Liang;WANG Cui-yan;BAI Lu;CHU Yan-fei;SHI He;GU Tie-jun;LIU Da-wei;BAO Xiao-hua(College of Life Sciences,Jilin Agricultural University,Changchun 130118,Jilin Province,China;不详)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2022年第7期853-857,共5页
Chinese Journal of Biologicals
基金
吉林省产业自主创新能力项目专项-蛋白质工程创新技术平台(2019C006)。
