施马伦贝格病毒N蛋白双单抗夹心ELISA检测方法的建立  

Establishment of double-monoclonal antibody sandwich ELISA assay for the detection of SBV N protein

作　　者：魏方 陈冬杰 邓俊花[1] 王晶晶 吴绍强[1] WEI Fang;CHEN Dongjie;DENG Junhua;WANG Jingjing;WU Shaoqiang(Chinese Academy of Inspection and Quarantine,Beijing,100176,China)

机构地区：[1]中国检验检疫科学研究院,北京100176

出　　处：《质量安全与检验检测》2023年第3期12-15,31,共5页QUALITY SAFETY INSPECTION AND TESTING

基　　金：中国检验检疫科学研究院基本科研业务费项目(2022JK11);“十四五”国家重点研发计划青年科学家项目(2022YFD1802000)。

摘　　要：为建立施马伦贝格病毒(Schmallenberg virus,SBV)的双抗体夹心ELISA方法,本研究利用原核表达系统纯化SBV N蛋白,并免疫小鼠制备抗SBV N蛋白单克隆抗体(mAb)。以制备的mAb 4D6作为捕获抗体,HRP标记的mAb 5D3经作为检测抗体,经条件优化,建立了基于SBV N蛋白的双单抗夹心ELISA检测方法。结果显示,捕获抗体和检测抗体的稀释度分别为1∶160000和1∶80000,重组SBV N蛋白浓度在3.9~125 ng/mL范围与OD450nm值呈现良好的线性关系,标准曲线为y=0.0224x+0.3896,R^(2)=0.9715。此方法检测下限为3.9 ng/mL,显示较高的敏感性。该方法检测AKAV、BTV和BVDV均为阴性,具有良好的特异性。因此,本研究建立的双单抗夹心ELISA方法为SBV快速诊断和N蛋白功能研究提供技术支撑。In order to establish a double antibody sandwich ELISA methed of Schmallenberg virus(SBV),the SBV N protein was expressed in prokaryotic expression system and as an immunogen to immunize mice for preparation of the monoclonal antibodies.The monoclonal antibodie(mAb)4D6 were used as the capture antibody,and the HRP-labeled mAb5D3 were used as the detection antibody.With optimization of the reaction conditions,a double-monoclonal antibody sanwich ELISA assay based on SBV N protein was established in this study.The results showed that the optimal dilution of the capture mAb 4D6 and HRP-labeled mAb 5D3 were 1:160000 and 1:80000,respectively.SBV N protein concentration ranging from 3.9 ng/mL to 125 ng/mL has a good linear relationship with OD450nm values,and the standard curve is y=0.0224x+0.3896,R^(2)=0.9715.The detected limit is 3.9 ng/mL,displaying the high sensitivity of the assay.There was no reaction with AKAV,BTV and BVDV,which indicated the good specifity of the ELISA method.These results showed that the established double-monoclonal antibody sandwich ELISA methed can be used for rapid diagnosis of SBV and provide technical support for SBV N protein function research.

关 键 词：施马伦贝格病毒 核衣壳蛋白 双单抗夹心ELISA 

分 类 号：S855.3[农业科学—临床兽医学]