摘要
利用口蹄疫病毒(Foot and mouth disease virus type O,FMDV-O)VP1特异性蛋白为抗原,杂交瘤细胞7E3分泌单克隆抗体,建立一种检测FMDV-O型的阻断酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)方法.对ELISA反应条件进行优化:抗原包被浓度为48.4 g/mL,单抗稀释度为28.0 g/mL,羊抗鼠酶标二抗(Sheep anti-mouse enzyme-conjugate secondary antibody,IgG-HRP)最佳稀释度为1 g/mL,底物液反应时间为37℃避光显色15 min.ELISA结果的判定标准为:血清阴断率<57%判为阳性,血清阻断率≥57%判为阴性.本研究建立的ELISA方法能与FMDV-O型阳性血清进行特异性反应,而与水泡性口炎病毒(Vesicular stomatitis virus,VSV),口蹄疫亚洲I型病毒(Foot and Mouth Disease virus type Asia1,FMDV-Asia1),羊痘病病毒(Sheep pox virus,QRF),口蹄疫A型病毒(Foot and mouth disease virus type A,FMDV-A),蓝舌病病毒(Bluetongue virus,BTV),小反刍兽疫病毒(peste des petits ruminants virus,PPRV),赤羽病病毒(Akabane virus,AKV)的阳性血清无交叉反应性.ELISA的重复性试验表明,批间和批内的变异系数均小于10%.比较试验结果表明,该ELISA的相对敏感性和特异性分别为88.9%和100%,与金迈诗口蹄疫O型液相阻断ELISA抗体检测试剂盒的符合率为93.75%.综上所述,本试验建立的口蹄疫病毒O型阻断ELISA抗体方法具有特异、稳定、灵敏的优点,为建立标准化的检测试剂盒奠定了基础.
A blocking Enzyme-linked immunosorbent assay(ELISA)for the detection of type O foot-and-mouth disease virus(FMDV-O)was established by using specific protein of VP1 group of FMDV-O as raw material to prepare antigen and hybridoma cell 7E3 monoclonal antibody to prepare detection antibody.The ELISA reaction conditions were optimized as follows:the concentration of antigen coating was 48.4 g/mL;the dilution of monoclonal antibody was 28.0 g/mL;the optimum dilution of commercial sheep anti-mouse enzyme-conjugated secondary antibody(IgG-HRP)was 1g/mL;and the reaction time of substrate solution was 15 min in the dark at 37℃.ELISA results were determined by the following criteria:serum blocking rate<57%was considered as positive,and serum blocking rate≥57%was considered as negative.The ELISA method established in this study can specifically react with the positive serum of FMDV-O,while has no cross-reaction with the positive serum of vesicular stomatitis virus(VSV),foot-and-mouth disease virus type Asia 1(FMDV-Asia 1),foot-and-mouth disease virus type A(FMDV-A),bluetongue virus(BTV),peste des petits ruminants virus(PPRV)and Akabane virus(AKV).The repeatability test of ELISA showed that the coefficient of variation between and within batches was less than 10%.The comparative test results showed that the relative sensitivity and specificity of the ELISA method established in this study were 88.9%and 100%respectively,and the coincidence rate was 93.75%with Jinmaishi FMDV-O liquid phase blocking ELISA antibody detection kit.In conclusion,the FMDV-O blocking ELISA method established in this study has the advantages of specificity,stability and sensitivity,which lays a solid foundation for the establishment of a standardized detection kit.
作者
张国春
李静
叶玲玲
罗倩敏
李瑶瑶
董仙兰
邹丰才
艾军
ZHANG Guo-Chun;LI Jing;YE Ling-Ling;LUO Qian-Min;LI Yao-Yao;DONG Xian-Lan;ZOU Feng-Cai;AI Jun(Yunnan Agricultural University,Kunming 650201;Technology Center of Kunming Customs,Kunming 650200)
出处
《中国口岸科学技术》
2022年第S01期51-56,共6页
China Port Science and Technology
基金
云南省技术创新人才项目(2016HB018)
云南农业大学兽医公共卫生省创新团队项目(202105AE160014)
云南省教育厅科学研究基金项目(2021J0116)
作者简介
第一作者:张国春(1999—),女,汉族,云南文山人,在读硕士,主要从事动物疫病检测技术研究,E-mail:1766492856@qq.com;通信作者:艾军(1977—),男,汉族,山西柳林人,博士,研究员,主要从事外来动物疫病检测技术研究,E-mail:52563122@qq.com
