口蹄疫病毒O型中和抗体检测固相阻断ELISA方法的建立  

Development of a solid-phase blocking ELISA for the detection of neutralizing antibodies against foot-and-mouth disease virus serotype O

作　　者：邢向川 李坤[1] 付元芳[1] 包慧芳[1] 李冬[1] 卢曾军[1] 刘在新[1] 曹轶梅[1] XING Xiangchuan;LI Kun;FU Yuanfang;BAO Huifang;LI Dong;LU Zengjun;LIU Zaixin;CAO Yimei(State Key Laboratory of Veterinary Etiological Biology,OIE/National Foot-and-Mouth Disease Reference Laboratory of China,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,Gansu,China;Tianjin Weite Biological Medicine Co.,Ltd.,Tianjin 300301,China)

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,国家口蹄疫参考实验室,甘肃兰州7300462 [2]天津威特生物医药有限公司,天津300301

出　　处：《微生物学报》2022年第11期4517-4528,共12页Acta Microbiologica Sinica

基　　金：国家重点研发计划(2021YFD1800300)。

摘　　要：[目的]为评价O型口蹄疫病毒(foot-and-mouth disease virus,FMDV)灭活疫苗免疫后中和抗体(neutralizing antibodies,NA)水平,建立检测中和抗体的固相阻断ELISA(neutralizing antibodies solid-phase blocking ELISA,NA-SPBE)方法。[方法]本研究以本实验室前期制备的FMDV广谱反应性牛源单克隆抗体E32作为捕获抗体,以生物素标记的FMDV O型型内广谱中和牛源单克隆抗体C4作为检测抗体,经过条件优化建立了检测FMDV O型中和抗体的固相阻断ELISA方法,并对该方法进行了敏感性、特异性、重复性、交叉反应性与病毒中和试验(virus neutralization test,VNT)相关性等试验。[结果]抗体E32最佳包被浓度为0.5μg/mL,O型FMDV灭活抗原最佳稀释浓度为0.25μg/mL,生物素标记抗体C4-Bio最佳工作浓度为0.06μg/mL,链霉亲和素-HRP的最佳稀释度为1:40000。以1.35 log10作为效价判定临界值时,敏感性和特异性分别为97.14%和98.84%。利用该方法分别检测FMDV A型、FMDV Asia1型、BVDV、PRRSV、CSFV、PPRV抗体阳性血清时,均为阴性,未出现交叉反应。该方法批内和批间重复试验的变异系数均<10%,表明其重复性较好。利用该方法与VNT分别对160份血清样品进行检测,二者的相关系数r为0.8075,P<0.0001,相关性显著。[结论]该方法可以检测FMDV O型中和抗体,为FMDV O型灭活疫苗免疫效果评价提供有力技术支撑。[Objective]To evaluate the level of neutralizing antibodies(NA)against foot-and-mouth disease virus(FMDV),a solid-phase blocking enzyme-linked immunosorbent assay(ELISA)based on bovine monoclonal antibodies was developed.[Methods]In this study,the bovine monoclonal antibody(mAb)E32 was used as the capture antibody and a biotinylated bovine mAb C4 as the detection antibody.Both mAbs had been produced in our laboratory.The mAb E32 is a cross-reactive antibody against FMDV and the mAb C4 is an intraserotype broadly neutralizing antibody against FMDV serotype O.With the two mAbs,a solid-phase blocking ELISA for detecting neutralizing antibody(NA-SPBE)against FMDV serotype O was developed.The sensitivity,specificity,repeatability,cross-reactivity,and correlation with virus neutralization test(VNT)of this assay were assessed.[Results]The optimum working concentrations of antibody E32,FMDV-inactivated antigen,and biotinylated C4 were 0.5μg/mL,0.25μg/mL and 0.06μg/mL,respectively,and the optimum dilution of streptavidin-HRP was 1:40000.When 1.35 log10 was used as the cut-off value,the sensitivity and specificity of the assay were determined as 97.14%and 98.84%,respectively.There was no cross reactivity with the antibodies specific to bovine viral diarrhea virus(BVDV),porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),peste des petits ruminant virus(PPRV),or FMDV serotypes A and Asia1.The intra-batch and inter-batch repeatability of the assay showed the coefficient of variation less than 10%.The detection of 160 serum samples demonstrated a correlation(r=0.8075,P<0.0001)between the titers obtained by NA-SPBE and VNT.[Conclusion]NA-SPBE can detect the neutralizing antibodies against FMDV serotype O and provides powerful technical support for evaluating the efficacy of FMDV serotype O-inactivated vaccine.

关 键 词：口蹄疫病毒 牛源单克隆抗体 中和抗体 固相阻断ELISA 

分 类 号：S852.65[农业科学—基础兽医学]