口蹄疫病毒O型全自动磁微粒CLIA抗体定量检测方法的建立  

Development of a Full-Automated Magnetic Particle Chemiluminescence Immunoassay Assay for Quantitative Detection of Antibodies Against Foot and Mouth Disease Virus Serotype O

作　　者：包艳芳 蒋韬 何莉[1] 吕律 李涛善 李昕 孙燕燕 杨光 魏婷 潘晓乐 林密 BAO YanFang;JIANG Tao;HE Li;LÜLü;LI TaoShan;LI Xin;SUN YanYan;YANG Guang;WEI Ting;PAN XiaoLe;LIN Mi(Lanzhou Shouyan Biotechnology Co.,Ltd,Lanzhou 730046;Key Laboratory of Veterinary Etiological Biology/National Foot-and Mouth Disease Reference Laboratory/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046)

机构地区：[1]兰州兽研生物科技有限公司,兰州730046 [2]中国农业科学院兰州兽医研究所/动物疫病防控全国重点实验室/国家口蹄疫参考实验室,兰州730046

出　　处：《中国农业科学》2024年第4期810-819,共10页Scientia Agricultura Sinica

基　　金：中央引导地方科技发展资金项目(23ZYQA295)。

摘　　要：【背景】口蹄疫(foot and mouth disease,FMD)是由口蹄疫病毒引起的一种急性、热性、烈性传染病,疫苗接种是预防临床口蹄疫的有效措施。免疫抗体水平监测则是评估疫苗免疫效果、制定免疫程序的重要依据,是免疫工作和疫情防控必不可少的关键环节,因此建立高效、快速、全自动的抗体检测方法具有重要意义。【目的】基于磁微粒(micromagnetic particles,MPs)化学发光免疫分析技术(CLIA),建立一种新型、全自动、可定量的口蹄疫病毒O型抗体检测方法,为口蹄疫免疫监测和疫情防控提供技术支撑。【方法】使用纳米材料磁微粒为固相载体和分离载体包被捕获抗体,用碱性磷酸酶(alkaline phosphatase,ALP)标记检测抗体,经过条件优化建立了一种新型磁微粒CLIA检测方法(magnetic particle chemiluminescence immunoassay,MP-CLIA)。本方法首先加入磁微粒-兔抗偶联物(magnetic particle-polyclonal antibodies,MPs-p Abs)、待测样本、口蹄疫病毒O型抗原,37℃孵育;再加入适量酶标抗体(ALP-p Abs),37℃孵育;最后加入化学发光底物AMPPD,检测相对发光强度(relative light unit,RLU)。研究通过检测标准品拟合标准曲线,并应用受试者工作特征曲线(receiver operating characteristic,ROC)确定检测方法的判定标准;利用质控样本进行方法学评价,同时检测田间样本,并和液相阻断ELISA(LPB-ELISA)比对,验证临床检测效果。【结果】优化后最佳反应条件为磁微粒浓度0.25 mg·m L^(-1)、口蹄疫病毒O型抗原1﹕1000稀释、酶标抗体1﹕2000稀释、加样量20μL。整个检测过程均在全自动化学发光免疫分析仪中完成,反应时间20 min,在抗体含量0—1280 U(效价0—1﹕2048)范围内标准曲线R2>0.99,可进行定量检测。该方法敏感性为94.66%;特异性为97.10%,检测口蹄疫A型、Asia I型血清型特异性为97.14%,与塞内卡病毒(SVV)、猪繁殖与呼吸综合征病毒(PRRSV)、牛流行热病毒(BEFV)、【Background】Foot-and-mouth disease(FMD)is an acute,febrile and virulent infectious disease caused by foot-and-mouth disease virus(FMDV),and vaccination is an effective measure to prevent the spread of FMD.The level of immune antibody monitoring is an important basis of evaluating the effectiveness of vaccine immunization,and developing immunization procedures.It is an indispensable step in the prevention and control of FMD.Therefore,to establish an efficient,rapid and fully automated antibody detection method is of great significance.【Objective】The aim of this study was to establish a novel,fully-automatic and quantitative antibody detection method for FMD virus type O based on the chemiluminescence immunoassay(CLIA)technology of magnetic particles(MPs),in order to provide the technical support for the immunization monitoring of FMD and epidemic prevention and control.【Method】In this study,the magnetic nanoparticles were used for solid-phase carriers and separation carriers to encapsulate the captured antibody,the detection antibodies was labelled by using alkaline phosphatase(ALP),and AMPPD was used for the luminescent substrate.Finally,a new magnetic particle Chemiluminescence CLIA method(MP-CLIA)was established after optimization of conditions.In this method,firstly,the magnetic particle-polyclonal antibodies(MPs-pAbs),the samples to be tested,and the antigen of FMDV type O were added to a reaction system and incubated at 37℃,then the appropriate amount of enzyme-labeled antibodies(ALP-pAbs)was added and incubated at 37℃,finally,the chemiluminescence substrate AMPPD was added to detect the relative light unit(RLU).In this study,a standard curve was fitted by testing the standards,and thereceiver operating characteristic(ROC)curve was applied to determine the determination criteria of the assay.The quality control samples were utilized for methodological evaluation,and the field samples were also tested and compared with the liquid phase blocking ELISA(LPB-ELISA)to validate the assay results.【

关 键 词：磁微粒化学发光免疫分析法 口蹄疫 磁微粒 全自动化 

分 类 号：S852.65[农业科学—基础兽医学]