CV-A5抗体制备及ELISA抗原检测方法的建立  

Generation of anti-CV-A5 polyclonal and monoclonal antibodies and establishment of quantitative antigen ELISA

作　　者：张小宇 靳卫平 王文辉 吴杰 卢佳 孟胜利 王泽鋆 申硕 ZHANG Xiao-Yu;JIN Wei-Ping;WANG Wen-Hui;WU Jie;LU Jia;MENG Sheng-Li;WANG Ze-Jun;SHEN Shuo(Wuhan Institute of Biological Products Co.,Ltd.,Laboratory 1 of Viral Vaccine,National Engineering Tech-nology Research Center of Combined Vaccines,Wuhan 430207,China)

机构地区：[1]武汉生物制品研究所有限责任公司病毒性疫苗研究一室,国家联合疫苗工程技术研究中心,武汉430207

出　　处：《中国免疫学杂志》2021年第11期1346-1351,共6页Chinese Journal of Immunology

基　　金：国家“重大新药创制”科技重大专项(2015ZX09102021);2017湖北省技术创新专项(2017ACA078)资助。

摘　　要：目的:制备柯萨奇病毒A组5型(CV-A5)多克隆抗体和单克隆抗体,建立CV-A5抗原定量ELISA检测方法,用于CV-A5疫苗研制中抗原定量及质量控制。方法:纯化后的CV-A5全病毒颗粒作为免疫原,制备兔多克隆抗体并免疫BALB/c小鼠,采用常规细胞融合技术制备、中和试验和ELISA法筛选获得CV-A5单克隆抗体。建立CV-A5抗原检测方法,确定线性范围,验证其准确度、精密度、稳定性、特异性;检测CV-A5病毒颗粒纯化过程样品抗原含量。结果:制备了高效价的CV-A5兔多克隆抗体及单克隆抗体并建立ELISA抗原检测法,检测范围为15.61000.0 ng/ml;高、中、低3个浓度样品准确度验证回收率为88.5%128.7%;重复性验证CV分别为1.3%、3.2%、1.2%;中间精密度验证CV分别为2.1%、2.2%和3.5%;耐用性验证回收率为97.4%127.6%;包被微孔板37℃放置3 d,样品回收率为101.7%106.9%;特异性验证结果显示抗原检测方法仅识别CV-A5抗原,与CV-A5以外的抗原均无交叉反应。结论:建立的CV-A5 ELISA抗原检测法可用于纯化过程样品的抗原检测,为含CVA5的手足口病(HFMD)多价疫苗的研制提供质量控制方法。Objective:To generate monoclonal and polyclonal antibodies against Coxsackievirus A5(CV-A5),and to establish a quantitative antigen ELISA for quality control in process development of CV-A5 vaccine preparation.Methods:Purified CV-A5 virions were used as immunogens to raise rabbit polyclonal antibodies and to immunize BALB/c mice,hybridomas secreting anti-CV-A5 monoclonal antibodies were obtained by conventional cell fusion technique,ELISA and neutralization test screening. Linear range of established CV-A5 ELISA was determined. Accuracy,precision,stability,and specificity were verified. Samples of purification processes of CV-A5 particles were used to determine antigen yields following each purification steps.Results:High-titers anti-CV-A5 polyclonal and monoclonal antibodies were generated. A quantitative ELISA for antigen detection was established. Detection range was15.6~1 000.0 ng/ml. Samples with high,medium and low concentrations were used to verified,whose recovery rates were 88.5%~128.7%. Repeatability verification results showed that CV were 1.3%,3.2%,1.2%;intermediate precision verification results showed that the CV were 2.1%,2.2%,3.5%;recovery rates were 97.4%~127.6%;coated microtiter plate was placed at 37°C for 3 d,and samples recovery rates were 101.7%~106.9%;specificity verification results showed that method only recognized CV-A5 and had no cross-reactivity with antigens other than CV-A5.Conclusion:Established CV-A5 quantitative ELISA can be applied to antigen detection of samples at different stages in purification process of CV-A5 antigen,which provides a quality control method for process development of multivalent HFMD vaccine containing CV-A5 component.

关 键 词：柯萨奇A组5型 多克隆抗体 单克隆抗体 双抗体夹心ELISA 抗原检测方法 

分 类 号：R392.11[医药卫生—免疫学]