CV-A4单克隆抗体制备及候选疫苗型特异性定量分析方法的建立和验证  

Preparation of CV-A4 monoclonal antibodies and establishment and validation of a type-specific quantitative assay for CV-A4 candidate vaccines

作　　者：田宇璇 汪梦俊 王文辉[1] 吴杰[1] 郭靖[1] 申硕[1] TIAN Yu-xuan;WANG Meng-jun;WANG Wen-hui;WU Jie;GUO Jing;SHEN Shuo(Wuhan Institution of Biological Product,Wuhan 430207,China)

机构地区：[1]武汉生物制品研究所,武汉430207

出　　处：《现代免疫学》2023年第1期8-15,共8页Current Immunology

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

摘　　要：为制备柯萨奇病毒A组4型(coxsackievirus A4,CV-A4)单克隆抗体并鉴定其生物学特性,同时为初步建立CV-A4抗原定量ELISA检测法,用于CV-A4候选疫苗研制中的抗原定量检测及质量控制,将纯化后的CV-A4全病毒颗粒免疫BALB/c小鼠,采用常规细胞融合技术、中和试验和ELISA获得CV-A4单克隆抗体;建立双抗体夹心ELISA检测法并进行方法学验证,用于检测手足口病(hand-foot-mouth disease,HFMD)多价疫苗中CV-A4抗原水平。结果显示,制备了型特异性CV-A4单克隆抗体并建立了双抗体夹心ELISA检测法,检测范围为62.5~4000.0 ng/mL;高、中、低3个浓度样本准确度验证,回收率为91.8%~121.9%;重复性验证,变异系数分别为3.4%、11.9%和8.6%;中间精密度验证,变异系数分别为1.8%、9.4%和6.5%;耐用性验证,回收率为97.3%~104.1%;包被微孔板37℃放置3 d,样本回收率为98.5%~119.2%;在特异度验证中,双抗体夹心ELISA检测法仅识别CV-A4抗原,与CV-A4以外的抗原均无交叉反应。由此,新建立的双抗体夹心ELISA检测法可用于纯化过程样本的CV-A4抗原检测,也可定量分析HFMD多价疫苗中CV-A4组分,这为多价疫苗的研制奠定了基础。This study aims to generate monoclonal antibodies,identify their biological characteristics,and establish a coxsackievirus A4(CV-A4)antigen quantitative ELISA assay for quality control in the development of CV-A4 candidate vaccines.Purified CV-A4 virions were used as immunogens to immunize BALB/c mice.Anti-CV-A4 monoclonal antibodies were obtained by conventional cell fusion technique,neutralization test,and ELISA.A double antibody sandwich ELISA was established,validated and used to detect the CV-A4 antigen of a multivalent candidate vaccine against hand-foot-mouth disease(HFMD).The results showed that type-specific anti-CV-A4 monoclonal antibodies were obtained and double antibody sandwich ELISA was established.The detection range was 62.5-4000.0 ng/mL.Samples at high,medium,and low concentrations were used to verify the accuracy and the recovery rates were 91.8%-121.9%.Results of repeatability verification showed that the coefficient of variations were 3.4%,11.9%,and 8.6%respectively.Results of intermediate precision verification showed that the coefficient of variations were 1.8%,9.4%,and 6.5%respectively.Results of durability verification showed that the recovery rates were 97.3%-104.1%.When coated microtiter plate was placed at 37°C for 3 d,samples recovery rates were 98.5%-119.2%.Results of specificity verification showed that detection was specific only to CV-A4 and had no cross-reactivity with other antigens tested.In summary,the validated double antibody sandwich ELISA can be used for CV-A4 antigen detection of samples in the purification process.In addition,the CV-A4 component in a multivalent HFMD vaccine can be quantitatively analyzed,thus laying the foundation for the development of multivalent vaccines.

关 键 词：柯萨奇病毒A组4型 单克隆抗体 双抗体夹心酶联免疫吸附试验 抗原检测 

分 类 号：R373.2[医药卫生—病原生物学]