不同合成方法对4种渔药人工抗原免疫 原性的影响

Effects of different synthesis methods on the immunogenicity of 4 kinds of fishery medicine artificial antigens

中文关键词:  碳二亚胺法  戊二醛法  重氮法  渔药  牛血清蛋白  鸡卵清蛋白  酶联免疫法

英文关键词:carbodiimide method  glutaraldehyde method  diazotization method  fishery medicine  bovine serum albumin  ovalbumin  enzyme-linked immunoadsordent assay

中文摘要:

      目的 建立4种常用渔药磺胺甲恶唑(sulfamethoxazole, SMZ)、新霉素(neomycin, NEO)、恩诺沙星(enrofloxacin, EF)和氟苯尼考(florfenico, FF)人工抗原合成及免疫原性的检测方法, 为其单克隆抗体制备及渔药残留快速检测奠定基础。方法 对4种渔药分别采用重氮化法、戊二醛法(GA)、碳二亚胺法(EDC)和混合酸酐法合成人工抗原。用紫外扫描法(UV)和聚丙烯酰胺凝胶电泳法(SDS-PAGE)对人工合成抗原进行鉴定, 并用ELISA法检测人工抗原的免疫原性。结果 重氮化法与GA法都能合成SMZ人工抗原, 重氮化法合成抗原的紫外峰值与电泳条带位移更明显, 抗体效价为1×104; EDC法与GA法均可合成NEO人工抗原, 但EDC法合成抗原的电泳条带位移更加明显, 且抗体效价为1×104; 混合酸酐法合成EF人工抗原失败, EDC法可合成EF人工抗原且合成抗原的紫外峰值与电泳条带位移明显, 抗体效价为1×104; GA法合成的FF人工抗原的紫外峰值与电泳条带位移明显, 抗体效价高达1×105。结论 SMZ人工抗原的合成采用重氮化法较好, FF人工抗原的合成采用戊二醛法较好, NEO与EF人工抗原的合成采用碳二亚胺法较好。

英文摘要:

      Objective To establish artificial antigen synthesis and immunogenicity test methods of sulfamethoxazole, neomycin, enrofloxacin and florfenico, and lay the foundation of preparation of their monoclonal antibody and rapid determination of fishery medicine residues. Methods Artificial antigens of 4 kinds of fishery medicine were compounded by diazotization method, glutaraldehyde method (GA), carbodiimide method (EDC) and mixed anhydride method, respectively, and authenticated by the method of ultraviolet spectroscopy (UV) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the immunogenicity was tested by indirect-ELISA method. Results SMZ artificial antigen could be compounded by diazotization method and GA method, but the UV and electrophoretogram of artificial antigen compounded by diazotization method were more obvious, and the antibody titer could reach 1×104. NEO artificial antigen could be compounded by EDC method and GA method, but the UV and electrophoretogram of artificial antigen compounded by EDC method were more obvious, and the antibody titer could reach 1×104. EF artificial antigen could be compounded by EDC method, mixed anhydride method was failed in compounding EF artificial antigen, the UV and electrophoretogram of artificial antigen compounded by EDC method were obvious, and the antibody titer can reach 1×104. The UV and electrophoretogram of FF artificial antigen compounded in GA method were obvious, and the antibody titer can reach 1×105. Conclusion Diazotization method was better for synthesis of SMZ artificial antigen, GA method was better for synthesis of FF artificial antigen, and EDC method was better for synthesis of NEO artificial antigen and EF artificial antigen.