猪伪狂犬病病毒gB蛋白单克隆抗体的制备及竞争ELISA检测方法的建立  

Preparation of Monoclonal Antibody Against gB Protein of Porcine Pseudorabies Virus and Development of a Competitive ELISA Detection Method

作　　者：杨大为 刘书怡 蔺雅婷 陈虎 张洪亮 李桂梅 YANG Dawei;LIU Shuyi;LIN Yating;CHEN Hu;ZHANG Hongliang;LI Guimei(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China;Novel Veterinary Pharmacy Innovation Center of Shandong Province,Qingdao 266109,China;Research Center for Engineering Technology in Veterinary Medicine and Veterinary Diagnostic Reagent of Shandong Province,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,青岛266109 [2]山东省新兽药创制协同创新中心,青岛266109 [3]山东省兽药诊断试剂工程技术研究中心,青岛266109

出　　处：《中国畜牧兽医》2024年第12期5439-5448,共10页China Animal Husbandry & Veterinary Medicine

基　　金：山东省重点研发计划(重大科技创新工程)项目(2023CXGC010705);山东省生猪产业技术体系(SDAIT-08-07)。

摘　　要：【目的】制备猪伪狂犬病病毒(Pseudorabies virus,PRV)gB蛋白单克隆抗体并建立竞争ELISA检测方法,为PRV的抗体检测及试剂盒开发提供参考。【方法】本研究使用PRV gB蛋白作为抗原免疫BALB/c小鼠,使用PEG1500将小鼠脾细胞与SP2/0细胞进行细胞融合,用间接ELISA方法筛选出阳性杂交瘤细胞,采用有限稀释法经过2轮亚克隆,将阳性杂交瘤细胞通过体内诱生法制备腹水,并通过饱和硫酸铵法对腹水进行纯化制备单克隆抗体,用单克隆抗体建立竞争ELISA检测方法。使用棋盘法对反应条件进行优化,对阴性血清进行检测,计算该方法的临界值,并进行特异性、灵敏性和重复性试验,最后与商品化试剂盒同时对临床血清样品进行检测,比较二者的符合率。【结果】获得2D6、2D8两株杂交瘤细胞均为IgG1型,轻链为κ链,建立的竞争ELISA方法单克隆抗体最佳稀释度为1∶400,蛋白最佳包被浓度为1μg/mL,待检血清最佳稀释浓度为1∶4,最佳封闭液为1%BSA,酶标二抗的最佳稀释度为1∶4000。与其他临床常见的猪病病原不发生交叉反应,具有良好的特异性。阳性血清稀释至1∶256仍为阳性,证明该方法灵敏性好。批内和批间的变异系数均<10%,证明建立的竞争ELISA方法具有良好的重复性。与商品化试剂盒比较,总体符合率达93.3%。【结论】本研究成功制备针对PRV gB蛋白的单克隆抗体,并应用单克隆抗体建立了PRV gB抗体竞争ELISA检测方法。【Objective】This study was aimed to prepare monoclonal antibodies against Porcine pseudorabies virus(PRV)gB protein and establish a competitive ELISA for detection of PRV antibody,provide reference for antibody detection of PRV and development of kits.【Method】In this study,BALB/c mice were immunized with PRV gB protein,and PEG1500 was used to facilitate cell fusion between mice spleen cells and SP2/0 cells.Positive hybridoma cells were screened by indirect ELISA.Through two rounds of sub-cloning using the limiting dilution method,positive hybridoma cells were induced in vivo to produce ascites,and monoclonal antibodies from the ascites were purified by ammonium sulfate precipitation.Furthermore,the monoclonal antibodies were used to develop a competitive ELISA.The chessboard method was used to optimize the reaction conditions and the negative sera were tested to calculate the cutoff value.The assay was evaluated and compared with commercial kits.【Result】Two strains of hybridoma cells,named as 2D6 and 2D8 were obtained.Both were IgG1 type withκ-chain light chain.The optimal dilution of monoclonal antibody of the established competitive ELISA method was 1∶400,the optimal coating concentration of protein was 1μg/mL,the optimal dilution of serum to be examined was 1∶4.The optimal blocking solution was 1%BSA,and the optimal dilution of enzyme-labeled secondary antibody was 1∶4000.It exhibited good specificity with no cross-reactivity with other porcine viruses.It remained positive when positive serum diluted to 1∶256,indicating that the method had high sensitivity.The coefficients of variation for both intra-assay and inter-assay were less than 10%,demonstrating that the established competitive ELISA method showed high repeatability.Compared with the commercialized kit,the consistency between the established ELISA and commercial test kit reached 93.3%.【Conclusion】In this study,monoclonal antibodies against the PRV gB protein were successfully prepared,and a PRV gB antibody competitive ELISA de

关 键 词：伪狂犬病病毒(PRV) gB蛋白 单克隆抗体 ELISA 

分 类 号：S852.651[农业科学—基础兽医学]