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

doi:10.16431/j.cnki.1671-7236.2022.02.030

摘要/Abstract

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1×10⁶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>10⁴; 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2⁹和2¹⁰, 腹水效价分别达10⁷和10⁸。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2 μg/mL浓度包被, 抗rFgCat L1多克隆抗体检测浓度为25 μg/mL, 酶标二抗稀释度为1∶4 000, 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0.625 μg/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。

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

Abstract: 【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, 1×10⁶ 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 10⁴.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 2⁹ and 2¹⁰ respectively, with ascites titers of 10⁷ and 10⁸.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 concentration of 2 μg/mL, the dilution concentration of anti-rFgCat L1 polyclonal antibody was 25 μg/mL, the dilution of Don-HRP-conjugated was 1∶4 000, 5% skimmed milk powder was selected as the blocking solution and the color development time was 25 min.The method was proved that could recognize the lowest antigen concentration of 0.625 μg/mL, also could specifically recognize antigen of Fasciola fasciatus.The constructed sandwich ELISA method was used for antigen detection of 47 dairy cow positive serum and 47 goat positive serum infective samples kept in the laboratory and the positive antigen rate were 72.3% and 78.7%, respectively.【Conclusion】 Anti-rFgCat L1 monoclonal antibody was successfully prepared and the double-sheet sandwich ELISA method for fascioliasis was constructed, which provided a good theoretical basis and material basis for the development of low-cost and rapid diagnostic kits.

Key words: Fasciala gigantica; cathepsin L1; monoclonal antibody; double antibody sandwich ELISA

中图分类号:

R446.61

王乐铭, 王瑜瑞, 曾子轩, 饶国顺, 吴正姣, 靳纬坤, 王冬英. 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[J]. 中国畜牧兽医, 2022, 49(2): 677-686.

WANG Leming, WANG Yurui, ZENG Zixuan, RAO Guoshun, WU Zhengjiao, JIN Weikun, WANG Dongying. Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 677-686.

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