重组肺炎球菌蛋白疫苗抗原含量双抗体夹心ELISA检测方法的建立、优化及验证  

Development,optimization and verification of double antibody sandwich ELISA for antigen content detection in recombinant pneumococcal protein vaccine

作　　者：沙芳芳 隋秀文 周朝东 朱婉玉 徐丽爽 韩强 朱涛 SHA Fangfang;SUI Xiuwen;ZHOU Chaodong;ZHU Wanyu;XU Lishuang;HAN Qiang;ZHU Tao(CanSino Biologics Inc,Tianjin 300301,China)

机构地区：[1]康希诺生物股份公司,天津300301 [2]天津市药品检验研究院,天津300070

出　　处：《中国生物制品学杂志》2025年第1期80-88,共9页Chinese Journal of Biologicals

基　　金：国家科技重大专项(2014ZX09102046)。

摘　　要：目的建立基于肺炎球菌表面蛋白A(pneumococcal surface protein A,PspA)的重组肺炎球菌蛋白疫苗中P3296、P5668和PRX13种抗原含量的双抗体夹心ELISA检测方法,并进行优化、验证和初步应用,以期为该疫苗的质量监测提供可靠的检测方法。方法用P5668、P3296和PRX1纯化蛋白免疫雄性新西兰大白兔,免疫血清经Protein A-Sephaorse 4B亲和层析纯化后,获得P5668、P3296和PRX1多克隆抗体,以其为包被抗体,HRP标记的相应单克隆抗体为酶标抗体建立抗原双抗体夹心ELISA检测方法。优化包被抗体浓度(3种多克隆抗体均稀释为2、4、8μg/mL)及酶标抗体稀释度(HRP标记的P5668单克隆抗体按1∶4000和1∶8000稀释、HRP标记的P3296单克隆抗体按1∶40000和1∶60000稀释、HRP标记的PRX1单克隆抗体按1∶12000和1∶24000稀释)、封闭液种类(1%BSA、1%鱼皮明胶、1%脱脂乳粉和1%酪蛋白)、稀释液种类(纯化水、1×PBS、2×PBS)、稀释液pH(6.4、7.4、8.4),并验证方法的线性范围、特异性、准确性、精密性、耐用性。分别采用建立的方法及Lowry法检测P5668、P3296、PRX1纯化蛋白(各20批),并分析2种方法检测结果的相关性。将3批P5668、P3296和PRX1混合疫苗经丙磺酸内盐解吸附后,采用建立的方法检测P3296、P5668和PRX1抗原含量。结果纯化的P5668、P3296和PRX1多克隆抗体蛋白浓度分别为1.27、2.20和1.53 mg/mL。P3296、P5668、PRX1多克隆抗体最佳包被浓度均为4μg/mL,HRP标记的P5668、P3296、PRX1单克隆抗体最佳稀释度分别为1∶4000、1∶60000、1∶12000;最佳封闭液为1%BSA,稀释液为1×PBS,稀释液pH为7.4。P5668、P3296和PRX13种参考品在3.125~100 ng/mL范围内,与A_(450)呈良好的线性关系,且R^(2)均>0.99;3种抗原高、中、低浓度加标样本回收率均在80%~120%范围内;3种抗原检测方法均可检出相应的特异性抗原,对其他2种抗原检测结果远低于实际含量或未检出;重复性及中间精密性验证CV�Objective To develop double antibody sandwich ELISA methods for the determination of P3296,P5668 and PRX1 antigen components in recombinant pneumococcal protein vaccine based on pneumococcal surface protein A(PspA),and to optimize,verify and preliminary apply it,in order to provide a reliable detection method for the quality monitoring of the vaccine.Methods The male New Zealand white rabbits were immunized with P5668,P3296 and PRX1 purified proteins.The immunized serum was purified by Protein A-Sephaorse 4B affinity chromatography,and P5668,P3296 and PRX1 polyclonal antibodies were obtained.Using the polyclonal antibodies as the coating antibodies and the corresponding HRP-labeled monoclonal antibodies as the enzyme-labeled antibodies,the double antibody sandwich ELISA methods were developed.The concentration of coating antibodies(all three polyclonal antibodies diluted to 2,4 and 8μg/mL),the dilution of enzyme-labeled antibodies(HRP-labeled P5668 monoclonal antibody diluted at 1∶4000 and 1∶8000,HRP-labeled P3296 monoclonal antibody diluted at 1∶40000 and 1∶60000,HRP-labeled PRX1 monoclonal antibody diluted at 1∶12000 and 1∶24000),the sealing liquid type(1%BSA,1%fish skin gelatin,1%skimmed milk powder and 1%casein),the diluent type(purified water,1×PBS,2×PBS),and the diluent pH(6.4,7.4,8.4)were optimized.The linear range,specificity,accuracy,precision,and robustness of the methods were verified.The developed methods and Lowry method were used to detect purified proteins of P5668,P3296 and PRX1(20 batches each),and the correlation between the results of the methods was analyzed.Three batches of P5668,P3296 and PRX1 mixed vaccines were desorbed by propanesulfonic acid internal salt,and the antigen contents of P3296,P5668 and PRX1 were detected by the developed methods.Results The protein concentrations of purified P5668,P3296 and PRX1 polyclonal antibodies were 1.27,2.20 and 1.53 mg/mL,respectively.The optimal coating concentration of P3296,P5668 and PRX1 polyclonal antibodies was all 4μg/mL,and th

关 键 词：重组肺炎球菌蛋白疫苗 肺炎球菌表面蛋白A 双抗体夹心ELISA法 抗原含量 

分 类 号：R392-33[医药卫生—免疫学]