O型口蹄疫Cathay拓扑型病毒单抗制备及双抗体夹心ELISA方法的初步建立

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中文摘要：本研究旨在纯化O型口蹄疫Cathay拓扑型病毒抗原,并制备单克隆抗体,为口蹄疫新型Cathay毒株的研究提供生物材料.PEG沉淀法纯化O型口蹄疫Cathay毒株抗原,免疫BALB/c小鼠,取脾细胞与SP2/0细胞融合制备杂交瘤细胞,用间接ELISA方法筛选阳性细胞,有限稀释法进行亚克隆,获得2株能够稳定分泌特异性针对O型口蹄疫Cathay株病毒单克隆抗体的杂交瘤细胞,分别命名为10E6与11C7.叠加试验表明,两株单抗的叠加率为49.45％,识别不同的抗原表位.间接ELISA和IFA试验显示,两株单抗与O型口蹄疫Cathay株病毒具有良好的反应性.单抗特异性检测结果表明,2株单抗均能特异性识别Cathay株口蹄疫病毒,不与其他口蹄疫病毒毒株交叉反应.抗体亚型鉴定结果显示,10E6单抗的轻链为Kappa链,11C7单抗的轻链为Lamda链,两株单抗重链类型均为IgG2a.结果表明,本研究成功制备了两株特异性结合O型口蹄疫Cathay拓扑型病毒的单克隆抗体,两株单抗均具有良好的反应性与特异性.应用两株单抗建立O型口蹄疫Cathay病毒的双抗体夹心ELISA检测方法,该ELISA检测方法特异性、重复性和敏感性良好,为O型口蹄疫Cathay毒株的快速诊断防控与研究建立了基础.

外文标题：Preparation of Monoclonal Antibody against Cathay Topotype of FMDV Type O and Development of Double Antibody Sandwich ELISA for Cathay Topotype of FMDV Type O

外文摘要：This study aimed to purify the cathay topotype of foot-and-mouth disease virus(FMDV)type O and prepare monoclonal antibody to provide biological materials for the study of novel cathay strain of foot-and-mouth disease.The antigen of cathay strain of FMDV type O was purified by PEG precipitation method and immunized BALB/c mice.Hybridoma cells were prepared by fusion of spleen cells and SP2/0 cells.Positive cells were screened by indirect ELISA and subcloned by limited dilution method.Two hybridoma cells,named 10E6 and 11C7,which were able to secrete monoclonal antibodies specifically against O-type FMDV Cathay strain were obtained.Additivity assay showed that the additivity rate of the two mAbs was 49.45％,and different epitopes were identified.Indirect ELISA and IFA showed that the two strains had good reactivity with O-type FMDV Cathay strain.The results of monoclonal antibody detection showed that the two monoclonal antibodies could specifically recognize Cathay FMDV strains and did not cross-react with other FMDV strains.The results of antibody subtype identification showed that the light chain of 10E6 monoclonal antibody was Kappa chain,the light chain of 11C7 monoclonal antibody was Lamda chain,and the heavy chain type of both strains was IgG2a.The results showed that two monoclonal antibodies specifically binding to O-type FMDV Cathay topological strain were successfully prepared in this study.Both monoclonal antibodies had good reactivity and specificity.Two monoclonal antibodies were used to establish a double antibody sandwich ELISA method for detecting O-type FMDV Cathay strain.The ELISA method had good specificity,repeatability and sensitivity,which laid a foundation for the rapid diagnosis,control and research of O-type foot and mouth disease virus Cathay strain.

外文关键词：

foot-and-mouth disease virus type OCathay topotypehybridoma cellsmonoclonal antibodyELISA

作者：

廖焕程、石正旺、罗俊聪、王婉莹、冯露、周静、张帆、石鑫泰、田宏

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作者单位：

中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,兰州 730000

关键词：

O型口蹄疫病毒 Cathay拓扑型杂交瘤细胞单克隆抗体 ELISA

基金：

2022甘肃省创新联合体国家重点研发计划项目中央高校基本科研业务费专项国家生猪技术创新中心中国农业科学院创新计划2023农业技术产业体系建设甘肃省科技重大专项计划

项目编号：

22ZD6NA0122021YFD1800300NCTIP-XD/C03CAAS-CSLPDC-202302CARS-3522ZD6NA001

出版年：

2024

DOI：

10.11843/j.issn.0366-6964.2024.09.025

畜牧兽医学报

中国畜牧兽医学会

畜牧兽医学报

CSTPCD北大核心

影响因子：0.729

ISSN：0366-6964

年,卷(期)：2024.55(9)