卵清蛋白多克隆抗体的制备及其双抗体夹心ELISA检测方法的建立  

Preparation of Ovalbumin Polyclonal Antibody and Establishment of Double Antibody Sandwich ELISA

作　　者：李妮 朱文勇[1] 宋绍辉[1] 钏鸿云 吴雅楠[1] 赵蕊蕊[1] 廖国阳[1] Li Ni;Zhu Wenyong;Song Shaohui(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Yunnan 650118,China)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所,昆明650000

出　　处：《医学研究杂志》2021年第12期55-59,共5页Journal of Medical Research

基　　金：中国医学科学院医学与健康科技创新工程项目(2020-I2M-2-014)。

摘　　要：目的建立卵清蛋白(ovalbumin,OVA)双抗体夹心ELISA检测方法并进行验证,用于定量检测鸡胚流感疫苗中的OVA含量。方法建立双抗体夹心酶联免疫法(enzyme-linked immunosorbent assay,ELISA),对该方法的反应条件进行优化,确定定量范围及检测下限,并对重复性、特异性和准确度进行验证。结果建立的方法最适包被抗体和酶标抗体浓度分别为2.5μg/ml和0.5μg/ml,最适封闭液配方为1%BSA+1%蔗糖。该方法的cut-off值为0.134,线性范围为0.313-20.000ng/ml,定量下限为0.078ng/ml。重复性好,板内变异系数为3.428%~25.953%,板间变异系数为5.375%~27.614%。特异性好,准确度高,检测结果与试剂盒检测结果符合率为91.43%~102.96%。稳定性好,预包被的酶标板冻存于-20℃,半年内检测样本变异系数为1.976%~14.409%。结论建立了快速、简便、低成本的OVA定量检测方法,可用于鸡胚流感疫苗中OVA定量检测。Objective To establish and verify a double antibody sandwich ELISA detection method for determination of ovalbumin in influenza vaccine.Methods A double antibody sandwich ELISA for ovalbumin was developed.The reaction conditions of this method were optimized,the quantitative range and the lower limit of detection were determined,and the repeatability,specificity and accuracy were verified.Results The optimal coating antibody and enzyme-labeled antibody concentrations of the established method were 2.5μg/ml and 0.5μg/ml,respectively,and the 1%BSA and 1%sucrose was used as blocking reagent.The Cut-off value of this method is 0.134,the linear range and detection lower limit of the method were 0.313-20.000ng/ml and 0.078ng/ml respectively.The interplate and intraplate coefficient of variation were 3.428%-25.953%and 5.375%-27.614%respectively,indicating a reliable repeatability.The compliance rate between this method and the kit is 91.43%-102.96%,revealing a high accuracy,and high specificity.The coefficient of variation of the sample tested by prepared plate was frozen and stored at-20℃within half a year was between 1.976%and 14.409%,indicating a reliable stability.Conclusion The developed method showed lower cost and convenience,which might be used for quantitative detection of ovalbumin in chicken embryo influenza vaccine.

关 键 词：卵清蛋白 多克隆抗体 双抗体夹心ELISA 

分 类 号：R392.1[医药卫生—免疫学]