轮状病毒抗原定量参比品的研制及抗原含量双抗体夹心ELISA的验证  

Development of quantitative reference and verification of double antibody sandwich ELISA method for rotavirus antigen

作　　者：宋菲菲 马莹 杨洋 张立杰 杜琳 邹强 SONG Feifei;MA Ying;YANG Yang;ZHANG Lijie;DU Lin;ZOU Qiang(Viral Vaccine R&D Center,Beijing Zhifei Luzhu Biopharmaceutical Co.,Lid.,Beijing 100176,China)

机构地区：[1]北京智飞绿竹生物制药有限公司病毒性疫苗研发中心,北京100176

出　　处：《微生物学免疫学进展》2024年第3期26-33,共8页Progress In Microbiology and Immunology

摘　　要：目的制备轮状病毒抗原定量参比品,建立检测轮状病毒抗原含量的双抗体夹心ELISA,并进行验证及初步应用。方法参照轮状病毒灭活疫苗生产工艺制备轮状病毒抗原定量参比品,以轮状病毒多克隆抗体为包被抗体,以轮状病毒单克隆抗体为检测抗体,建立双抗体夹心ELISA,对建立的方法进行线性、准确度、精密度、专属性、预包板稳定性及参比品稳定性验证;将验证后的该方法初步应用于轮状病毒灭活疫苗制备工艺的监控及产品质量分析。结果制备的轮状病毒抗原参比品纯度>95%,2~8℃贮存稳定性不低于12个月。建立的双抗体夹心ELISA,标准曲线线性范围3.75~120.00 U/mL;包被抗体和检测抗体的质量浓度分别为2μg/mL和100 ng/mL;各质量浓度抗原回收率均在90.5%~109.8%;不同质量浓度样品检测结果的实验内CV在1.9%~6.3%,实验间CV在4.4%~7.2%;与Vero细胞蛋白、DMEM细胞培养液、新生牛血清、EV71抗原、CA16抗原无交叉反应;预包板于2~8℃放置21 d,抗原回收率均在91.0%~110.2%,CV均<10%;将该方法应用于各工艺阶段抗原回收率监测,病毒澄清、超滤及第一步柱层析抗原回收率均在83.6%~93.1%;第二步柱层析和原液制备工序抗原回收率分别为62.2%和70.7%。结论制备的轮状病毒抗原内参品纯度、稳定性均符合用于轮状病毒抗原定量标定的要求;建立的轮状病毒抗原含量双抗体夹心ELISA检测方法具有良好的线性、准确度、精密度和专属性。用该方法进行轮状病毒抗原含量检测可作为轮状病毒灭活疫苗制备过程中各工艺质量控制的重要指标。Objective To prepare a quantitative reference for rotavirus antigen and establish a double antibody sandwich ELISA assay for the detection of rotavirus antigen content followed by validation and preliminary application.Methods The production process of inactivated rotavirus vaccine(IRV)was used as a reference to prepare a quantitative reference standard for rotavirus antigen.A double antibody sandwich ELISA method was established using rotavirus polyclonal antibodies as the coating antibody and rotavirus monoclonal antibody as the detection antibody.The established method was subjected to validation for linearity,accuracy,precision,specificity,pre-coated plate stability,and reference standard stability.The validated method was then applied for monitoring the production process and analyzing the quality of IRV.Results The prepared reference rotavirus antigen exhibits a purity exceeding 95%and demonstrates storage stability at 2-8℃for at least 12 months,suitable for quantitative calibration of rotavirus antigen.The double-antibody sandwich ELISA established in this study exhibited a linear range of 3.75 to 120.00 U/mL for the standard curve.The concentration of the coating antibody and detection antibody were 2μg/mL and 100 ng/mL respectively.The antigen recovery rates at different concentrations were within the range of 90.5%to 109.8%.The experimental intraassay coefficient of variation(CV)for the detection of samples with different concentrations ranged from 1.9%to 6.3%,the inter-assay CV ranged from 4.4%to 7.2%.Specificity is confirmed by the absence of cross-reactivity with Vero cell protein,DMEM medium,BSA,EV71,and CA16.This method was applied to monitor the antigen recovery rates at various stages of the process.The virus clarification,ultrafiltration,and the first step of column chromatography showed antigen recovery rates ranging from 83.6%to 93.1%.The second step of column chromatography and the raw material preparation process exhibited antigen recovery rates of 62.2%and 70.7%.The pre-coated plates rem

关 键 词：轮状病毒 抗原 标准品 双抗体夹心法 酶联免疫吸附测定 验证 质量控制 

分 类 号：R373.2[医药卫生—病原生物学]