抗大片形吸虫Cat L1单克隆抗体的制备及其双抗体夹心ELISA检测方法的建立  

Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala giganticaand Establishment of Double Antibody Sandwich ELISA

作　　者：王乐铭 王瑜瑞 曾子轩 饶国顺 吴正姣 靳纬坤 王冬英[1] WANG Leming;WANG Yurui;ZENG Zixuan;RAO Guoshun;WU Zhengjiao;JIN Weikun;WANG Dongying(College of Animal Science and Technology,Guangxi University, Nanning 530004,China)

机构地区：[1]广西大学动物科学技术学院,南宁530004

出　　处：《中国畜牧兽医》2022年第2期677-686,共10页China Animal Husbandry & Veterinary Medicine

基　　金：广西创新驱动发展专项(桂科AA17204057);广西自然科学基金(2019GXNSFAA245013);崇左市科技项目(崇科FA2019006)。

摘　　要：【目的】制备抗大片形吸虫组织蛋白酶L1(rFgCat L1)特异性单克隆抗体,并构建其双抗体夹心ELISA检测方法。【方法】用1 mg/mL rFgCat L1蛋白分4次对5只BALB/c小鼠进行免疫,分离小鼠脾细胞,与SP2/0细胞融合构建杂交瘤细胞,筛选强阳性杂交瘤细胞株,每只小鼠腹腔注射1×10^(6)个细胞制备单克隆抗体。ELISA法检测抗体效价及抗原表位,Western blotting法鉴定抗体亚型和特异性;结合抗rFgCat L1多克隆抗体构建双抗体夹心ELISA检测方法,并检测其敏感性和特异性;用建立的方法对20份阴性血清及阳性血清对照进行检测,筛选其阴阳临界值,并用47份山羊阳性血清及47份奶牛阳性血清对所构建的双抗体夹心ELISA方法进行验证。【结果】免疫后,4只小鼠血清中抗体效价均>10^(4);取小鼠脾细胞与SP2/0融合后,共筛选到8株阳性杂交瘤细胞株,其中5D5和7G6为可稳定分泌抗体的强阳性株,抗体效价分别达2^(9)和2^(10),腹水效价分别达10^(7)和10^(8)。经Western blotting鉴定2株抗体均为IgG1型,轻链为Kappa型,均可特异性结合大片形吸虫排泄-分泌产物(excretory-secretory product,ESP)。由于2株抗体识别相同抗原位点,对比2株单抗的抗体效价,选择以7G6作为捕获抗体,抗rFgCat L1多克隆抗体作检测抗体构建双抗体夹心ELISA法。双抗体夹心ELISA法条件为:7G6以2μg/mL浓度包被,抗rFgCat L1多克隆抗体检测浓度为25μg/mL,酶标二抗稀释度为1∶4000,5%脱脂奶粉封闭,显色时间为25 min。经验证,该方法可识别的最低抗原浓度为0.625μg/mL,并且可特异性识别大片形吸虫抗原。利用构建的双抗体夹心ELISA方法对阳性的奶牛和山羊血清样本进行抗原检测,抗原检出率分别为72.3%及78.7%。【结论】本研究成功制备抗rFgCat L1单克隆抗体并构建大片形吸虫病双抗体夹心ELISA检测方法,结果可为研发低成本、快速诊断试剂盒提供良好的理论依据及物质基础。【Objective】This study was intend to obtain cathepsin L1(rFgCat L1)specific monoclonal antibody and construct the double antibody sandwich ELISA.【Method】Five BALB/c mice were immunized with 1 mg/mL rFgCat L1 protein for four times.Mouse splenocytes were isolated and fused with SP2/0 cells to construct hybridoma cells.Strong positive hybridoma cell lines were screened,1×10^(6) cells were injected intraperitoneally per mouse to prepare monoclonal antibodies.Antibody titer and antigenic epitope were detected using ELISA method,antibody subtype and specificity were identified using Western blotting method.The double antibody sandwich ELISA was constructed by combining the anti-rFgCat L1 polyclonal antibody,and its sensitivity and specificity were tested.The positive and negative critical value was screened by 20 negative sera with positive control,and the constructed double antibody sandwich ELISA was verified by 47 goat positive sera and 47 dairy cow positive sera.【Result】After immunization,the antibody titers in serum of 4 mice were all more than 10^(4).After isolated mouse with the highest immune response spleen cells were fused with SP2/0 cells total of 8 of them were positive cell lines were obtained after selective culture.5D5 and 7G6 were identified as strong positive strains with stable antibody secretion.After multiple subcloning screens and subcultures,the antibodies secreted in the cell supernatant were stable,with titers of 2^(9) and 2^(10) respectively,with ascites titers of 10^(7) and 10^(8).Western blotting and antibody subtype identification kits identified that the two antibodies were IgG1 type and the light chain was kappa type,both of which could specifically bind FgESP.According to the same antigen site was recognized by the two kinds of antibodies,the antigen titer of the two monoclonal antibodies were comparied,7G6 was used as the coating antibody,and anti-rFgCat L1 was used as the enzyme-labeled secondary antibody.The optimized condition of method was that 7G6 was coated at a concen

关 键 词：大片形吸虫 组织蛋白酶L1 单克隆抗体 双抗体夹心ELISA 

分 类 号：R446.61[医药卫生—诊断学]