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目的制备抗尼罗罗非鱼血清免疫球蛋白的单克隆抗体,并对其免疫学特性进行鉴定,运用它们监测嗜水气单胞菌免疫尼罗罗非鱼血清抗体水平。方法采用重组蛋白A亲和层析法纯化尼罗罗非鱼血清免疫球蛋白,纯化蛋白经SDS-PAGE检测后免疫Balb/c小鼠,取免疫鼠脾细胞与Sp2/0细胞融合;用ELISA法对杂交瘤进行筛选以及测定单抗的特性和效价,Western blot和叠加ELISA分析单抗的抗原结合表位;灭活嗜水气单胞菌免疫尼罗罗非鱼,用单抗建立ELISA检测体系监测血清特异性抗体水平。结果 SDS-PAGE电泳条件下血清免疫球蛋白重链和轻链的相对分子质量分别约为77 000和27 000;共获得4株抗罗非鱼血清Ig的单抗,分别命名为3D9、9D4、8G3和7B7,它们的细胞上清ELISA效价为1∶3 200～1∶6 400,抗体Ig亚类均为IgG1;4株单抗都能特异识别罗非鱼免疫球蛋白,而与鲫、草鱼、南方鲇、斑点叉尾鮰、鲈鱼血清无任何交叉反应。3D9识别变性条件下的尼罗罗非鱼Ig重链,9D4、8G3和7B7为轻链特异性。初次免疫后1～15 d尼罗罗非鱼血清中嗜水气单胞菌特异性抗体水平显著上升,第3次免疫后3周抗体水平达到峰值。结论成功制备抗尼罗罗非鱼血清免疫球蛋白单克隆抗体,为尼罗罗非鱼免疫球蛋白的结构分析、免疫应答水平监测和病原诊断等研究提供有力工具。
Objective To prepare anti-Nile tilapia serum immunoglobulin monoclonal antibodies, and immunological characteristics were identified, the use of them to monitor Aeromonas hydrophila immune Nile tilapia serum antibody levels. Methods Serum immunoglobulin of Nile tilapia was purified by recombinant protein A affinity chromatography. Balb / c mice were immunized with SDS-PAGE and splenocytes of immunized mice were fused with Sp2 / 0 cells. The hybridoma was screened and the characteristics and potency of McAb were determined. Western blot and superposition ELISA were used to analyze the antigen-binding epitope of monoclonal antibody. Inactivated A. hydrophila was inactivated by Aeromonas hydrophila Detection system to monitor serum-specific antibody levels. Results The molecular weights of heavy chain and heavy chain of serum immunoglobulin under SDS-PAGE electrophoresis were about 77 000 and 27 000, respectively. A total of four monoclonal antibodies against IgA of tilapia were obtained and named as 3D9, 9D4, 8G3 and 7B7, their cell supernatant ELISA titers were 1: 300 ~ 1: 400, the antibody Ig subclasses were IgG1; 4 monoclonal antibodies were able to specifically identify tilapia immunoglobulin, and crucian carp, Grass carp, southern catfish, Channel catfish, sea bass serum without any cross-reaction. 3D9 recognizes the Nile tilapia Ig heavy chain under denaturing conditions, 9D4, 8G3 and 7B7 are light chain specific. The level of Aeromonas hydrophila-specific antibody in the serum of Nile tilapia increased significantly from 1 to 15 days after the first immunization, and the antibody level peaked at 3 weeks after the third immunization. Conclusion The preparation of monoclonal antibodies against tilapia anti-nile tilapia serum successfully provided a powerful tool for the structural analysis, immune response monitoring and pathogen diagnosis of nile tilapia immunoglobulin.