| 基于单克隆抗体的O型口蹄疫病毒抗体固相竞争ELISA检测方法的建立 |
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| 引用本文： | 许智强,李敏杰,刘彦玲,周国辉,于力.基于单克隆抗体的O型口蹄疫病毒抗体固相竞争ELISA检测方法的建立[J].中国预防兽医学报,2020(2):145-149. |
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| 作者姓名： | 许智强 李敏杰 刘彦玲 周国辉 于力 |
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| 作者单位： | 中国农业科学院哈尔滨兽医研究所牛羊传染病研究创新团队 |
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| 基金项目： | 国家重点研发计划(2016YFD0501505)。 |
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| 摘 要： | 为建立评价O型口蹄疫病毒(FMDV)疫苗免疫水平的方法,本研究以单克隆抗体(MAb)3D9为捕获抗体,以HRP标记的MAb 8E8作为检测MAb,经过条件优化建立了基于MAb的检测O型FMDV抗体的固相竞争ELISA(SPCE)方法。对该方法进行了特异性、敏感性、重复性试验。结果显示,MAb 3D9的最佳稀释度为1:25 000,灭活O型FMDV抗原的最佳稀释浓度为1:3,HRP标记的MAb 8E8的最佳稀释度为15 000,当血清1:32稀释时,检测的临界值确定为45%。该方法分别检测A型FMDV抗体阳性参考血清以及牛冠状病毒、牛轮状病毒以及猪繁殖与呼吸障碍综合征病毒、猪圆环病毒、猪瘟病毒的标准阳性血清,检测结果均为阴性,未出现交叉反应。经检测,当阳性标准血清的抗体稀释度在1:512时,该方法仍具有较好的敏感性;批内和批间重复性试验的变异系数均小于10%,表明其重复性较好。并将该方法与液相阻断ELISA(LPBE)方法和病毒中和试验(VNT)的相关性进行了比较,结果显示该方法与LPBE和VNT的相关性分别为0.896和0.923。本研究为国内评价O型FMDV疫苗免疫水平建立了一种新的方法。
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| 关 键 词： | 口蹄疫病毒 单克隆抗体 固相竞争ELISA |
Development of solid phase competition ELISA for detection of O-type foot-and-mouth disease virus antibodies based on monoclonal antibody |
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| XU Zhi-qiang,LI Min-jie,LIU Yan-ling,ZHOU Guo-hui,YU Li.Development of solid phase competition ELISA for detection of O-type foot-and-mouth disease virus antibodies based on monoclonal antibody[J].Chinese Journal of Preventive Veterinary Medicine,2020(2):145-149. |
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| Authors: | XU Zhi-qiang LI Min-jie LIU Yan-ling ZHOU Guo-hui YU Li |
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| Institution: | (Harbin Veterinary Research Institute,CAAS,Harbin 150069,China) |
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| Abstract: | In order to detect antibodies against foot-and-mouth disease virus(FMDV), the monoclonal antibody(MAb) 3D9 was used as the capture antibody, and the HRP-labeled MAb 8E8 was used as the detection antibody. After several conditions were optimized, the critical value of the detection was established. A solid phase competition ELISA(SPCE) method for the detecting of type O FMDV antibodies based on monoclonal antibodies. Specific, sensitive, and reproducible tests were performed on this method.The correlation between this method and the liquid phase blocking ELISA(LPBE) method and the virus neutralization test(VNT)was compared. The results showed that the optimal dilution of MAb 3D9 was 1:25 000, the optimal dilution of inactivated type O FMDV antigen was 1:3, and the optimal dilution of MAb 8E8 was 1:5 000. When the serum was diluted at 1:32, the cut-off value of the assay was determined to be 45%. The method was used to detect reference positive serum of type A FMDV, bovine coronavirus, bovine rotavirus, porcine reproductive and respiratory syndrome virus, porcine circovirus 2, and classical swine fever virus, respectively. The test results were all negative and no cross-reaction occurred. After testing, when the dilution of the positive standard serum is 1:512, the method still has good sensitivity;the coefficient of variation of the intra-assay and inter-assay repeated tests is less than 10%, indicating that the repeatability is better. The correlation between this method and VNT and LPBE was 0.923 and 0.896, respectively. This study established a new method for the detection of domestic type O FMDV antibodies in China. |
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| Keywords: | foot-and-mouth disease virus monoclonal antibody solid phase competition ELISA |
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