EV71抗原ELISA定量检测方法的建立  

Development of A Quantitative ELISA Method for EV71 Antigen

作　　者：贾卉[1] 蔡芳[2] 高强[2] 张建三[2] 井申荣[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650224 [2]北京科兴生物制品有限公司研发中心,北京100085

出　　处：《中国生物制品学杂志》2010年第1期87-90,共4页Chinese Journal of Biologicals

摘　　要：目的建立EV71抗原双抗体夹心ELISA定量检测方法,用于EV71灭活疫苗生产过程中抗原含量的检测。方法采用EV71全病毒免疫,制备抗EV71单克隆抗体及多克隆抗体,以抗EV71多克隆抗体作为包被抗体,经HRP标记的抗EV71单克隆抗体作为酶标抗体,建立双抗体夹心ELISA法,检测样品中EV71抗原含量,并对该方法进行验证及应用。结果所建立的方法线性范围为5～80U/ml,R2=0.994,线性关系良好,定量限度为5U/ml;检测不同浓度的EV71抗原内部参考品,回收率为88%～119.0%;变异系数均小于15%;与甲肝病毒收获液、乙脑病毒灭活液、H5N1流感病毒裂解疫苗原液、小牛血清、人白蛋白、Vero细胞、MEM培养基及CoxA16收获液均无交叉反应;检测EV71灭活疫苗原液纯化过程样品的抗原含量,能有效反映抗原纯化趋势;检测铝吸附疫苗成品解离后抗原含量,准确性较高。结论已建立了EV71抗原双抗体夹心ELISA定量检测方法,该方法线性好,特异性强,灵敏度高,准确性及重复性良好,可用于EV71疫苗生产过程中及终产品的抗原定量检测。Objective To develop a double antibody sandwich ELISA method for quantitative determination of EV71 antigen during production of inactivated EV71 vaccine.Methods Monoclonal and polyclonal antibodies against EV71 antigen were prepared by immunizing rabbits with complete EV71.A double antibody sandwich ELISA method was developed using polyclonal antibody as coating antibody and HRP-labeled monoclonal antibody as enzyme-labeled antibody and used for determination of EV71 antigen content in test samples,then verified and applied.Results The linear range and R2 value of the developed ELISA method were 5 ～ 80 U /ml and 0.994 respectively,indicating good linearity.The quantitation limit of the developed method was 5 U /ml.The recovery rate of internal reference of EV71 antigen at various concentrations was 88% ～ 119.0%,with a variation coefficient of less than 15%.No cross reaction with harvested hepatitis A virus liquid,inactivated Japanese encephalitis virus liquid,influenza H5N1 virus cleavage vaccine,calf serum,human albumin,Vero cells,MEM or harvested CoxA16 liquid was observeed.The determination result of EV71 antigen content in test samples of bulk of inactivated EV71 vaccine during purification reflected the tendency of antigen purification.The developed method showed high accuracy in determination of antigen content in dissociated final product of EV71 vaccine adsorbed onto aluminium salt.Conclusion A double antibody sandwich ELISA method for quantitative determination of EV71 antigen was developed,which showed good linearity and high specificity,sensitivity,accuracy and reproducibility and might be used for quantitative determination of antigen in EV71 vaccine during production and in final product.

关 键 词：EV71抗原 双抗体夹心ELISA 验证 

分 类 号：R373.2[医药卫生—病原生物学] R392.33[医药卫生—基础医学]