中国兽医科学2025，Vol.55Issue(4)：484-491,8.DOI:10.16656/j.issn.1673-4696.2025.0059
猪日本乙型脑炎病毒E蛋白抗体磁微粒化学发光法的建立
Establishment of a magnetic particle chemiluminescence method for antibody to E protein of Japanes encephalitis virus
摘要
Abstract
In order to provide a quicker and more convenient serological detection method for Japanes encephalitis virus(JEV)detection,a magnetic particle chemiluminescence immunoassay(CLIA)for Japanes encephalitis virus E protein(JEV-E)antibody was established in this experiment.The magnetic particles were coated with prokaryotically expressed JEV-E as antigen,and the detection system was composed consisted of the set up of serum to be examined and alkaline phosphatase-labeled IgG antibody to quantitatively detect the antibody content of JEV in porcine serum.The results showed that after optimizing the coupling conditions,the optimal buffer pH was 5.0,the optimal protein encapsulation amount was 30 μg/mL,5％BSA was used as a sealing agent,and the most suitable concentration of immuno-magnetic bead preservation solution was 1 mg/mL.After optimizing the reaction conditions,the dilution of alkaline phosphatase antibody was selected to be 1∶20 000,the reaction time of serum was 5 min,that of alkaline phosphatase was 10 min,and that of substrate was 7 min.The critical point of the magnetic particle chemiluminescence method is 24.99 U.The plotting of the ROC showed a sensitivity of 99.5％and a speci-ficity of 97.5％.No cross-reactivity with positive serum antibodies to porcine poliovirus(PPV),porcine foot-and-mouth disease O virus(FMDV-O),porcine circovirus 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),porcine Streptococcus(SS),and Haemophilus parvovirus(HPs).The inter-and intra-batch coefficients were less than 10％.The sensitivity was not significantly different from that of the commercialized kit.The compliance rate with the commercialized kit was 98.27％.The method is simple to operate and the test procedure is convenient and fast,which can provide new technical support for the rapid diagnosis of JEV.关键词
猪日本乙型脑炎病毒/E蛋白/磁微粒/化学发光法Key words
Japanes encephalitis virus/E protein/magnetic particle/chemiluminescence分类
农业科技引用本文复制引用
孙菁晗,向国庆,宋帅,温肖会,吕殿红,连聪,顾有方,罗胜军..猪日本乙型脑炎病毒E蛋白抗体磁微粒化学发光法的建立[J].中国兽医科学,2025,55(4):484-491,8.基金项目
广东省农业科学院动物卫生研究所青年科技人才培育项目(PY2024010) （PY2024010）
广东省科技计划项目(2021B1212050021) （2021B1212050021）
