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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1×10~4;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1×10~4;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1×10~4;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1×10~5。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。
OBJECTIVE To establish the synthetic and immunostimulatory activities of four commonly used antifungal agents, sulfamethoxazole (SMZ), neomycin (NEO), enrofloxacin (EF) and florfenico (FF) The detection of the original nature of the monoclonal antibodies for the preparation and rapid determination of residual drugs to lay the foundation. Methods The artificial antigens were synthesized by diazotization method, glutaraldehyde method (GA), carbodiimide method (EDC) and mixed acid anhydride method. The synthetic antigen was identified by ultraviolet (UV) and polyacrylamide gel electrophoresis (SDS-PAGE), and the immunogenicity of the artificial antigen was detected by ELISA. Results Both diazotization and GA methods could synthesize SMZ artificial antigen. The diazotization of the diazotized antigen was more obvious with the shift of electrophoresis band. The antibody titer was 1 × 10 ~ 4. The EDC method and GA method could both synthesize NEO artificial antigen, but EDC method of antigen synthesis electrophoresis band shift more obvious, and the antibody titer of 1 × 10 ~ 4; synthetic anhydride synthetic EF artificial antigen failure, EDC method can be synthesized EF artificial antigen and synthetic antigen peak UV The band of electrophoresis band was significantly shifted with the antibody titer of 1 × 10 ~ 4. The UV peak value and electrophoresis band shift of FF artificial antigen synthesized by GA method were obvious. The titer of antibody reached 1 × 10 ~ 5. Conclusion The synthesis of SMZ artificial antigen is better by diazotization. The synthesis of FF artificial antigen is better by glutaraldehyde method. The synthesis of NEO and EF artificial antigen is better by carbodiimide method.