重组Omicron BA.4/5-Delta株新冠疫苗体外相对效力检测方法的建立及验证  

Development and verification of in vitro relative potency detection method for recombinant Omicron BA.4/5-Delta SARS-CoV-2 vaccine

作　　者：刘莹 李静静 赵永杰 冯静静 毛慧芳 刘晓雅 LIU Ying;LI Jingjing;ZHAO Yongjie;FENG Jingjing;MAO Huifang;LIU Xiaoya(R&D of Anhui Zhifei Longcom Biopharmaceutical Co.,Ltd.,Hefei 230031,Anhui Province,China)

机构地区：[1]安徽智飞龙科马生物制药有限公司研发中心,安徽合肥230031

出　　处：《中国生物制品学杂志》2025年第1期61-66,共6页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2021YFC2302601)。

摘　　要：目的建立重组Omicron BA.4/5-Delta株新冠疫苗体外相对效力的检测方法,并进行验证,以期为替代体内效力检测奠定基础。方法以人源单克隆抗体GH4作为包被抗体,HRP标记的CB6人源单克隆抗体作为酶标抗体的双抗体夹心ELISA法为基础,确定疫苗解吸附方法;再以该解吸附方法结合双抗体夹心ELISA法建立体外相对效力检测方法。并验证方法的线性范围、专属性、准确性、精密性、耐用性及定量限。采用建立的方法检测3批供试品重组Omicron BA.4/5-Delta株新冠疫苗的体外相对效力。结果确定解吸附方法为:将疫苗与处理液(1.25 mL 20%二乙醇胺,0.20 mL 10%Triton X-100,8.55 mL PBS)按等体积分数混合,于25℃解离30 min,解吸附率可达95%以上。疫苗参考品在1~26 ng/mL浓度范围内,与A_(450)呈良好的线性关系,线性方程为log(y)=1.447 log(x)-1.643,R^(2)为0.998;可特异性检测疫苗参考品的体外相对效力;90000、50000和20000 ng/mL浓度疫苗参比品检测结果的回收率均在80%~120%范围内;重复性及中间精密性验证相对标准偏差(relative standard deviation,RSD)均<20%;解离条件、检测体系孵育时间和显色时间发生微小变动时,检测结果不受影响;定量限为0.2。批号为J202301002、J202301003、J202301004供试品的体外相对效力分别为1.0,1.0和0.8,RSD为11%。结论建立的用于检测体外相对效力的方法具有良好的准确性、精密性、专属性和耐用性,可用于重组Omicron BA.4/5-Delta株新冠疫苗体外相对效力的检测。Objective To develop and verify an in vitro relative potency detection method for recombinant Omicron BA.4/5-Delta SARS-CoV-2 vaccine,so as to lay a foundation for using in vitro relative potency test in place of in vivo potency test.Methods Based on a double antibody sandwich ELISA with human monoclonal antibody GH4 as coating antibody and HRPlabeled CB6 human monoclonal antibody(CB6-HRP)as enzyme-labeled antibody,the desorption method of vaccine was determined.Then,the desorption method was combined with double antibody sandwich ELISA to develop a relative potency detection method in vitro.The linear range,specificity,accuracy,precision,robustness and limit of quantitation(LOQ)of the method were verified.The developed method was used to detect the in vitro relative potency of three batches of recombinant Omicron BA.4/5-Delta vaccines.Results The desorption method was determined as follows:the vaccine was mixed with the treatment solution(1.25 mL 20%diethanolamine,0.20 mL 10%Triton X-100,8.55 mL PBS)in equal volume fraction,and dissociated at 25℃for 30 min,and the desorption rate was over 95%.In the concentration range of 1-26 ng/mL,the reference vaccine showed a good linear relationship with A_(450),and the linear equation was log(y)=1.447 log(x)-1.643,with the R^(2) of 0.998.The in vitro relative potency of reference vaccine was specifically detected.The recovery rates of results of 90000,50000 and 20000 ng/mL vaccines were in the range of 80%-120%.The relative standard deviations(RSDs)of repeatability and intermediate precision verification were both less than 20%.The slight change of dissociation conditions,incubation time and color development time of the detection system showed no effect on the detection results.The LOQ was 0.2.The in vitro relative potency of J202301002,J202301003 and J202301004 samples was 1.0,1.0 and 0.8,respectively,with an RSD of 11%.Conclusion The detection method developed for in vitro relative potency has good accuracy,precision,specificity and robustness,and can be used for the

关 键 词：重组Omicron BA.4/5-Delta株疫苗 解吸附 双抗体夹心ELISA法 体外相对效力 

分 类 号：Q533[生物学—生物化学]