检测水痘-带状疱疹病毒糖蛋白水平双抗体夹心ELISA法的建立和验证  

Establishment and validation of double antibody sandwich ELISA for determination of varicella zoster virus glycoprotein level

作　　者：朱琨莹 郭世杰[2] 袁若森 潘东 张春 陈晓旭 衣延明 杨阳 郑柏松 姜春来[4] ZHU Kunying;GUO Shijie;YUAN Ruosen;PAN Dong;ZHANG Chun;CHEN Xiaoxu;YI Yanming;YANG Yang;ZHENG Baisong;JIANG Chunlai(Institute of AIDS and Virology Research,First Hospital,Jilin University,Changchun 130021,China;Department of Neonatology,First Hospital,Jilin University,Changchun 130021,China;Changchun Baike Biotechnology Co.LTD,Changchun 130012,China;Department of Microbial Immunology,School of Life Science,Jilin University,Changchun 130012,China)

机构地区：[1]吉林大学第一医院艾滋病与病毒研究所,吉林长春130021 [2]吉林大学第一医院新生儿科,吉林长春130021 [3]长春百克生物科技股份公司,吉林长春130012 [4]吉林大学生命科学学院微生物免疫系,吉林长春130012

出　　处：《吉林大学学报（医学版）》2020年第6期1315-1323,共9页Journal of Jilin University：Medicine Edition

基　　金：科技部国家科技重大专项基金资助课题(2018ZX10302104-001-010);吉林省卫健委科研项目资助课题(20161J065);吉林省分子病毒学重点实验室科研项目资助课题(20102209)。

摘　　要：目的:建立可快速检测水痘-带状疱疹病毒(VZV)糖蛋白抗原水平的双抗体夹心ELISA法,并验证其特异性、线性、准确性和精密性。方法:采用抗VZV糖蛋白基因工程抗体建立双抗体夹心ELISA法,检测VZV糖蛋白抗原水平。优化抗体组合、抗体工作浓度、包被条件、封闭条件、酶标抗体反应条件和显色条件,并对建立的方法进行特异性、线性、准确性及精密性验证。结果:捕获抗体VZV-mAb-03和酶标抗体VZV-mAb-HRP-02的组合为最优双抗组合,其最适浓度分别为450.0和0.1μg·L^-1,最适包被条件为0.05 mol·L^-1 Tris-HCl(pH值为8.7±0.1)在4℃条件下包被过夜(18 h),最适封闭条件为1%牛血清白蛋白(BSA)在37℃条件下封闭2 h,最适样品稀释液为磷酸盐吐温缓冲液(PBST),抗原夹心与酶标抗体最适反应条件均为37℃、1 h,最适显色条件为37℃、15 min。经验证,该方法与其他人用疫苗及辅料成分无交叉反应;线性范围为500~2600 PFU·mL^-1,回归系数(R2)>0.95;批内回收率82.2%~115.7%,变异系数(CV)<10.2%;批间回收率80.5%~118.6%,板内CV<17.4%,板间CV<14.9%。结论:建立了一种基于检测双抗体夹心的VZV糖蛋白抗原水平的ELISA检测方法,符合定量检测需要,可用于VZV疫苗的研发和生产过程中的抗原水平检测。Objective:To establish a double antibody sandwich ELISA for rapid determination of varicella zoster virus(VZV)glycoprotein level,and to validate its specificity,linearity,accuracy,and precision.Methods:Double antibody sandwich ELISA was established with the recombinant antibodies against VZV glycoprotein and used for the determination of VZV glycoprotein level.The antibody combination,antibody concentration,coating conditions,blocking conditions,enzyme-labled antibody reaction conditions,and chromogenic condition were optimizated in the experiment,and the established method was validated for the specificity,linearity,accuracy,and precision.Results:The optimal combination of the double antibody was the group of VZV-mAb-03 as the capture antibody at a concentration of 450.0μg·L^-1 and VZV-mAb-HRP-02 as the enzyme-labeled antibody at a concentration of 0.1μg·L^-1;the optimal buffer for antibody coating was 0.05 mol·L^-1 Tris-HCl(pH=8.7±0.1),while the optimal temperature and time for coating were 4℃and overnight(18 h)respectively;the bovine serum albumin(BSA)at a concentration of 1%was served as the blocking agent,while the optimal temperature and time for blocking were 37℃and 2 h,respectively;the optimal reaction time of VZV and enzyme-labeled second antibody with PBST were both 1 h at the temperature of 37℃,and the optimal temperature and time for color reaction were 37℃and 15 min,respectively.It was proved that this method had no cross reactions with the other vaccines or ingredients.The quantitation scope was 500-2600 PFU·mL^-1,regression coefficient(R2)>0.95;the in-batch recovery rate was 82.2%-115.7%,and the in-batch coefficient of variable(CV)<10.2%;the batch-to-batch recovery was 80.5%-118.6%,the CV in validation for inplate<17.4%and the CV inter-plate precisions<14.9%.Conclusion:Double antibody sandwich ELISA suitable for determination of VZV glycoprotein level is established,which meets the requirements for quantitative determination,and might be used to determine the VZV antigen level during

关 键 词：水痘-带状疱疹病毒 糖蛋白 双抗体夹心 酶联免疫吸附试验 变异系数 

分 类 号：R373.9[医药卫生—病原生物学]