中国兽医科学2025，Vol.55Issue(7)：898-904,7.DOI:10.16656/j.issn.1673-4696.2025.0135
赤羽病病毒血清抗体间接ELISA检测方法的建立
Establishment of indirect ELISA detection method for serum antibodies against Akabane virus
摘要
Abstract
To establish an indirect ELISA method for the detection of serum antibodies to Akabane virus(AKAV),a prokaryotic expression vector for AKAV nucleocapsid(N)protein was constructed,and the prokaryotic expression of recombinant N protein was used as the coating antigen to establish the indi-rect ELISA detection method.The results showed that the N protein was expressed mainly as inclusion bod-ies with a size of 26 kDa.The established indirect ELISA method and the optimized detection conditions were as follows:the mass concentration of coating antigen was 1.00 mg/L,the condition of antigen coat-ing was incubated at 37 ℃ for 1 h;the serum dilution ratio was 1∶400,and the incubation condition was at 37 ℃ for 45 min;the HRP-labeled secondary antibody was incubated at 37 ℃ for 45 min;and substrate was incubated at 37℃ for 5 min in the dark.The value for the positive determination of this ELISA method was P/N=1.687.This indirect ELISA has no cross-reactivity with common ruminant pathogens positive sera,and the sensitivity was 1∶2 560,with intra-batch coefficient of variation less than 5.00％and inter-batch coefficient of variation less than 10.00％.The indirect ELISA method for serum antibody against AKAV established in this experiment can be applied to the detection of large number of samples in clinic,which can provide a theoretical basis for the early detection of AKAV.关键词
赤羽病/核衣壳(N)蛋白/原核表达/间接ELISA/血清抗体Key words
Akabane disease/nucleocapsid(N)protein/prokaryotic expression/indirect ELISA/serum antibody分类
农业科技引用本文复制引用
朱日宁,鞠安琪,王成宇,张芳毓,王永志..赤羽病病毒血清抗体间接ELISA检测方法的建立[J].中国兽医科学,2025,55(7):898-904,7.基金项目
吉林省农业科技创新工程项目(CXGC2022RCB005) （CXGC2022RCB005）
吉林省科技发展计划吉林省肉牛产业化发展重大科技专项(YDZJ202203CGZH050) （YDZJ202203CGZH050）
