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

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 the development and production of VZV vaccine.