摘要
为了建立一种赤羽病病毒(AKAV)血清抗体间接ELISA检测方法,构建AKAV核衣壳(N)蛋白原核表达载体,以原核表达的重组AKAV N蛋白为包被抗原,建立了间接ELISA检测方法。结果表明:N蛋白主要以包涵体形式表达,大小为26 k Da。建立的间接ELISA方法及优化后的最佳检测条件为:抗原包被质量浓度1.00 mg/L,包被条件为37℃孵育1 h;血清稀释比例为1∶400,37℃孵育45 min;HRP酶标二抗37℃孵育45 min;底物反应条件为37℃避光5 min;阳性判定临界值为P/N=1.687。该间接ELISA检测方法具有较好的特异性,与常见的反刍动物病原阳性血清不发生交叉反应,敏感性达到1∶2560,批内变异系数小于5.00%,批间变异系数小于10.00%。建立的AKAV血清抗体间接ELISA方法可以应用于赤羽病临床大量样本检测,为AKAV的早期检测奠定了理论基础。
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 indirect ELISA detection method.The results showed that the N protein was expressed mainly as inclusion bodies 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.o0 mg/L,the condition of antigen coating 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 37C 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 1:2560,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.
作者
朱日宁
鞠安琪
王成宇
张芳毓
王永志
ZHU Rining;JU Anqi;WANG Chengyu;ZHANG Fangyu;WANG Yongzhi(Jilin Academy of A gricultural Sciences(Northeast A gricultural Research Center of China),Changchun 130033,China)
出处
《中国兽医科学》
北大核心
2025年第7期898-904,共7页
Chinese Veterinary Science
基金
吉林省农业科技创新工程项目(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)。
