摘要
旨在建立猪繁殖与呼吸综合征病毒(PRRSV)多表位融合蛋白(MEFP)间接ELISA抗体检测方法并验证其应用效果。利用融合表达的PRRSV囊膜蛋白MEFP作为包被抗原,通过优化抗原包被浓度、血清稀释度和二抗稀释度等条件,建立检测PRRSV抗体的间接ELISA方法(MEFP-ELISA)。结果:该方法最佳抗原包被浓度为200 ng/孔,最佳血清稀释度为1∶100,最佳酶标二抗稀释度为1∶500;具有较高的特异性,与猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)等病原抗体无交叉反应;批内和批间变异系数均低于10%,表现出良好的重复性;用该方法与商品化PRRSV囊膜蛋白抗体ELISA试剂盒同时检测447份临床血清,结果显示,总符合率达92.39%,Kappa系数为0.8219;针对疫苗免疫猪的抗体动态监测结果表明,2种方法的抗体变化趋势较为一致;利用该方法检测湖南省13个核心种猪场的650份临床血清,其抗体总阳性率为34.62%,结合PRRSV N蛋白抗体ELISA试剂盒检测结果,综合分析了猪群的PRRSV感染及免疫状况。综上,本研究建立的MEFP-ELISA方法适用于PRRSV血清学检测,具有良好的临床应用前景。
This study was to establish an indirect ELISA method for detecting porcine reproductive and respiratory syndrome virus(PRRSV)antibodies using a multi-epitope fusion protein(MEFP)of PRRSV and validate the application of the method.The PRRSV MEFP was used as the coating antigen,and an indirect ELISA method(MEFP-ELISA)was developed by optimizing key parameters,including antigen coating concentration,serum dilution ratio,and secondary antibody dilution ratio.Here are the research results.The optimal conditions were determined as follows:antigen coating concentration of 200 ng/well,serum dilution of 1∶100,and enzyme-labeled secondary antibody dilution of 1∶500.The method exhibited high specificity,with no cross-reactivity observed with antibodies against porcine circovirus type 2(PCV2),classical swine fever virus(CSFV),or pseudorabies virus(PRV).Both the intra-assay and inter-assay coefficients of variation were below 10%,indicating good reproducibility.Comparative testing of 447 clinical serum samples using the MEFPELISA and a commercial PRRSV envelope protein ELISA kit showed an overall agreement rate of 92.39%and a Kappa coefficient of 0.8219.Furthermore,dynamic monitoring of antibody levels in vaccinated pigs revealed consistent trends between the two methods.When applied to 650 clinical serum samples from 13 core pig farms in Hunan Province,the MEFP-ELISA detected an overall antibody positive rate of 34.62%.Combined with the results from a PRRSV N protein ELISA kit,this method allows for comprehensive analysis of PRRSV infection and immunization status in pig herds.The MEFP-ELISA method established in this study is suitable for PRRSV serological detection and possesses promising potential for clinical application.
作者
陈志雄
勾明郗
范杰
唐小明
张坤
刘甜甜
李欣
赵文龙
李润成
葛猛
CHEN Zhixiong;GOU Mingxi;FAN Jie;TANG Xiaoming;ZHANG Kun;LIU Tiantian;LI Xin;ZHAO Wenlong;LI Runcheng;GE Meng(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Hunan Province Animal Disease Prevention and Control Center,Changsha 410014,China)
出处
《畜牧与兽医》
北大核心
2026年第1期71-78,共8页
Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金面上项目(32072871)
湖南省重点研发计划项目(2023NK2017)。
关键词
猪繁殖与呼吸综合征病毒
多表位融合蛋白
抗体
血清学检测
porcine reproductive and respiratory syndrome virus
multi-epitope fusion protein
antibody
serological testing
