卵清蛋白双抗体夹心ELISA检测方法的建立及验证  

Development and verification of double antibody sandwich ELISA for determination of ovalbumin

作　　者：李妮 朱文勇[1] 宋绍辉[1] 马磊[1] 高菁霞[1] 廖国阳[1] 李卫东[1] LI Ni;ZHU Wenyong;SONG Shaohui;MA Lei;GAO Jingxia;LIAO Guoyang;LI Weidong(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650118,Yunnan Province,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,云南昆明650118

出　　处：《中国生物制品学杂志》2023年第7期850-854,861,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81860281);中国医学科学院医学与健康科技创新工程(2020-l2M-2-014);中国医学科学院医学科技创新项目(2016-l2M-3-026)。

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,以用于流感疫苗中OVA含量的测定。方法 以兔抗OVA多克隆抗体作为包被抗体,HRP标记的兔抗OVA多克隆抗体作为检测抗体建立OVA双抗体夹心ELISA检测方法。对包被抗体浓度(2、1、0.5、0.25μg/mL)、酶标抗体浓度(0.5、0.25、0.125μg/mL)、封闭液(1%BSA、2%BSA、1%BSA+1%蔗糖、2%BSA+2%蔗糖,以未封闭为对照)进行优化,并确定Cut-off值作为判断标准。对方法的线性范围及检测下限、特异性、重复性和准确度进行验证。结果 该方法的最适检测条件为:包被抗体浓度1μg/mL,酶标抗体浓度0.25μg/mL;封闭液为2%BSA+2%蔗糖;Cut-off值为0.051 66。该方法线性范围为5~0.313 ng/mL,检测下限为0.078 ng/mL;与流感病毒、牛血清白蛋白(bovine serum albumin,BSA)、Vero细胞上清液等均不发生反应;板内变异系数(CV)在2.562%~13.887%之间,板间CV在4.000%~16.497%之间;该方法检测结果与德国SERAMUN OVA定量检测试剂盒检测结果的符合率在93.79%~107.05%之间。结论 建立的OVA双抗体夹心ELISA方法具有良好的特异性、重复性、线性范围及准确度,且操作简便、成本低,可用于OVA的定量检测。Objective To develop and verify a double antibody sandwich ELISA detection method for the determination of ovalbumin(OVA),in order to determine the OVA content in influenza vaccines.Methods The rabbit anti-OVA polyclonal antibody was used as coating antibody and HRP labeled rabbit anti-OVA polyclonal antibody as detection antibody to develop a double antibody sandwich ELISA for OVA.The antibody concentration(2,1,0.5 and 0.25 pg/mL) for coating,enzyme-labeled antibody concentration(0.5,0.25 and 0.125 μg/mL),and kinds of blocking reagent(blocking with1% BSA,blocking with 2% BSA,blocking with 1% BSA and 1% sucrose,and blocking with 2% BSA and 2% sucrose,using nonblocking as control) were optimized,and the Cut-off value was determined as the judgment standard.The developed method was verified for the linear range and detection limit,specificity,repeatability and accuracy.Results The optimum detection conditions were as follows:the concentration of coating antibody was 1 μg/mL,the concentration of enzyme-labeled antibody was 0.25 μg/mL;The blocking reagent was 2% BSA and 2% sucrose;The Cut-off value was 0.051 66.The linear range of the method was 5~0.313 ng/mL,and the detection limit was 0.078 ng/mL;It did not react with influenza virus,bovine serum albumin(BSA) and Vero cell supernatant;The intraplate and interplate coefficient of variation(CV) were between 2.562%~13.887% and 4.000%~16.497% respectively;The coincidence rate between the results of this method and the Germany SERAMUN OVA quantitative test kit ranged from 93.79% to 107.05%.Conclusion The developed OVA double antibody sandwich ELISA has good specificity,repeatability,linear range and accuracy with convenience and lower cost,which might be used for the quantitative detection of OVA.

关 键 词：卵清蛋白 鸡胚 双抗体夹心ELISA 

分 类 号：R392.1[医药卫生—免疫学]