甲型肝炎病毒抗原含量双抗体夹心ELISA检测方法的建立及验证  

Development and Validation of Double Antibody Sandwich ELISA for Determination of Hepatitis A Virus Antigen Content

作　　者：李冬琦 高旭哲 潘皓 于艳 苏文全 LI Dongqi;GAO Xuzhe;PAN Hao;YU Yan;SU Wenquan(Cheng Da Biotechnology(Benxi)Co.,Ltd.,Benxi 117000,China;Liaoning Cheng Da Biotechnology Co.,Ltd.,Shenyang 110000,China)

机构地区：[1]成大生物(本溪)有限公司,辽宁本溪117000 [2]辽宁成大生物股份有限公司,辽宁沈阳110000

出　　处：《生物化工》2023年第1期12-17,22,共7页Biological Chemical Engineering

摘　　要：目的:建立快速检测甲型肝炎病毒(HAV)抗原含量的双抗体夹心ELISA方法,并进行验证及初步应用。方法:用HAV多克隆抗体和HAV单克隆抗体酶结合物建立双抗体夹心ELISA法,检测HAV抗原含量。对建立的方法进行特异性、线性(标准曲线)、准确度、精密度、灵敏度验证,对不同生产厂家生产的HAV疫苗(人二倍体细胞)与不同工艺阶段的HAV疫苗(人二倍体细胞)中间产品进行适用性检测。结果:HAV多克隆抗体的最佳包被浓度为8μg/mL,酶标抗体最佳稀释度为1∶10 000,封闭剂为1%BSA。该方法与其他人用疫苗和辅料成分无交叉反应;线性范围0.546 875~17.500 000 IU/mL,R^(2)> 0.99;准确度验证回收率80.1%~123.7%;精密度验证变异系数<8.3%。检测不同厂家的HAV疫苗(人二倍体细胞)和不同工艺阶段的HAV疫苗(人二倍体细胞)中间产品呈良好的剂量依赖效应。结论:建立了适用于HAV抗原含量检测的双抗体夹心ELISA方法,符合定量检测的需求,可用于不同毒株生产的HAV疫苗(人二倍体细胞)检测,可用于甲肝病毒收获液、浓缩液、纯化液、灭活液及原液等不同工艺阶段样品检测。Objective:To develop,validate and preliminarily apply a double antibody sandwich ELISA for rapid determination of hepatitis A virus(HAV)antigen content.Methods:The double antibody sandwich ELISA is developed with HAV polyclonal antibodies and hepatitis a virus monoclonal antibody enzyme conjugate used for the determination of HAV antigen content.The developed method is validated for specificity,linearity(standard curve),accuracy,precision and sensitivity.The HAV(human diploid cells)produced by different manufacturers from different virus strains as well as the vaccine samples at different steps of production procedure are determined by the method to evaluate the suitability.Results:The optimal coated concentration of HAV polyclonal antibodies is 8μg/mL,the optimal dilutions of enzyme-labeled antibody is 1∶10000,and the optimal blocking agent is 1%BSA.There are no cross reactions with other vaccines or subsidiary materials.The linear range of the developed method is 0.546875~17.500000 IU/mL,with a R2 value of more than 0.99.The recovery rate in validation for accuracy is 80.1%~123.7%.The coefficient of variation in validation for precisions in the batch and between the batch are less than 8.3%.Dose-dependent effects are observed by the developed method in determination of HAV from different manufacturers and samples at different steps of production procedure.Conclusions:A double antibody sandwich ELISA suitable for HAV antigen content is developed,which meet the requirements for quantitative determination,and may be used for the HAV vaccines prepared from different virus strains as well as the samples at different steps of production procedure,such as HAV harvest solution,HAV concentrate,HAV purification,HAV inactivated solution and HAV bulk.

关 键 词：甲型肝炎病毒 定量检测 双抗体夹心ELISA法 

分 类 号：R512.61[医药卫生—内科学] R446.6[医药卫生—临床医学]