禽腺病毒4型Fiber-2蛋白单克隆抗体的制备及双抗体夹心ELISA检测方法的建立  

Preparation of monoclonal antibody against fowl adenovirus serotype 4 Fiber-2 protein and establishment of the double antibody sandwich ELISA method

作　　者：刘文健 王帅雯 吉梅 刘萌 汤智辉 张硕 宋素泉[1] 闫丽萍 LIU Wenjian;WANG Shuaiwen;JI Mei;LIU Meng;TANG Zhihui;ZHANG Shuo;SONG Suquan;YAN Liping(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Zhonghai Biopharmaceutical(Taizhou)Co.,Ltd.,Taizhou 225316,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]中海生物制药(泰州)有限公司,江苏泰州225316

出　　处：《畜牧与兽医》2024年第6期101-109,共9页Animal Husbandry & Veterinary Medicine

基　　金：泰州市“接榜挂帅”重点科技项目(JBGS202103)。

摘　　要：旨在制备禽腺病毒4型(FAdV-4)Fiber-2蛋白的单克隆抗体并建立Fiber-2蛋白的定量检测方法。本研究将原核表达的Fiber-2蛋白免疫BALB/c雌鼠,通过细胞融合技术制备杂交瘤细胞,并通过免疫印迹、间接免疫荧光和抗体相加试验筛选杂交瘤细胞株;以制备的单克隆抗体为捕获抗体和酶标检测抗体,通过优化捕获抗体包被浓度和酶标抗体稀释度等条件,建立特异性检测Fiber-2蛋白的双抗体夹心ELISA方法。结果:本研究成功筛选到3株能够稳定传代的杂交瘤细胞株1H2、2A9和3E1,其分泌的抗体均与FAdV-4全病毒具有良好的反应性,可识别不同抗原表位;进一步发现,以1H2为捕获抗体、2A9为酶标检测抗体,能够建立检测Fiber-2蛋白的ELISA方法;该方法具有良好的重复性,批内重复及批间重复试验变异系数均小于10%;敏感性高,Fiber-2蛋白检测限为0.078 ng/μL;特异性好,不与FAdV-4的其他结构蛋白及鸭腺病毒3型的Fiber-2蛋白发生反应。综上,本研究成功制备了3株Fiber-2蛋白单克隆抗体,并建立了定量检测Fiber-2蛋白的双抗体夹心ELISA方法,为后续FAdV-4基础研究和Fiber-2亚单位疫苗研究奠定了物质基础。This research aimed to prepare a monoclonal antibody for FAdV-4 Fiber 2 protein and to establish a specific quantitative detec⁃tion method for Fiber-2 protein.FAdV-4 Fiber-2 protein expressed in a prokaryotic system was firstly used to immunize 6-week-old BALB/c female mice,and spleen cell fusion was performed to prepare hybridoma cells.The prepared hybridoma cells were further screened in this study via western blotting assay,indirect immunofluorescence assay,and antibody addition experiments.Next by optimizing a series of conditions such as the concentration of the captured antibody coating and the dilution of the enzyme-labeled antibody,a dual-antibody sand⁃wich ELISA method capable of specifically detecting the concentration of Fiber-2 protein could be established.Finally,the prepared mono⁃clonal antibody was used as the capture antibody and the enzyme-labeled detection antibody in this detection method.The results showed that three hybridoma cell lines named 1H2,2A9 and 3E1 were successfully prepared in this study,and the monoclonal antibodies secreted by them reacted well with FAdV-4 and could recognize different antigenic epitopes.Further studies revealed that an ELISA method for the detec⁃tion of Fiber-2 protein could be established using 1H 2 as the capture antibody and 2A9 as the enzyme-labeled detection antibody.The double antibody sandwich ELISA method prepared based on the above monoclonal antibodies possessed good repeatability,and the coefficient of variation of repeated experiments within and between batches was below 10%.At the same time,the detection limit of Fiber-2 protein in this method was only 0.078 ng/μL,and it did not react with other structural proteins of FAdV-4 and Fiber-2 proteins of DAdV-3.Con⁃clusively,three monoclonal antibodies against Fiber-2 protein recognizing different epitopes were successfully prepared in this study,and a double-antibody sandwich ELISA method was successfully established to quantitatively detect the concentration of fiber-2 protein,which would ser

关 键 词：禽腺病毒血清4型 单克隆抗体 双抗体夹心ELISA 

分 类 号：S852.4[农业科学—基础兽医学]