碳二亚胺法制备阿莫西林人工抗原及其鉴定

Preparation and Identification of Amoxicillin Artificial Antigen by EDC Method

合成、鉴定了阿莫西林(amoxicillin,AMO)人工抗原,并通过动物免疫法生产了亲和力高、特异性好的鼠源AMO多克隆抗血清。采用碳二亚胺(EDC)法将AMO分别与载体蛋白BSA和OVA偶联,合成完全免疫抗原AMO-BSA和检测抗原AMO-OVA,经紫外分光光度法和SDS-PAGE以及动物免疫进行鉴定。结果表明,偶联后的紫外吸收峰与BSA和AMO相比都发生了一定的位移,AMO-BSA在276nm处出现最大吸收峰,BSA的泳动速度大于AMO-BSA。免疫后获得的3只小鼠多抗血清,通过间接竞争ELISA测定,效价可达1×10-4以上,1号小鼠半数抑制浓度(IC50)为573.75ng/mL,敏感性较好。AMO完全人工抗原的合成以及鼠源多克隆抗体血清的制备,为AMO单克隆抗体的制备奠定了基础。

To synthesize the artificial antigen of AMO and obtain its mouse polyclonal antiserum,the immunogen AMO-BSA and coating antigen AMO-OVA were synthesized using EDC method and identified by ultraviolet scanning and SDS-PAGE.BALB/c mice were immunized with the synthesized antigens and the polyclonal antiserum was determined by indirect and blocking ELISA.The results showed that after conjugation,the ultraviolet absorption peak of AMO-BSA appeared at 276 nm,indicating that certain displacement occurred comparing with the ultraviolet absorption peaks of both AMO and BSA.The electropharetic mobility of BSA was observed bigger than that of AMO-BSA.Indirect ELISA showed that the antiserum titres of all the three immunized BALB/c mice were above 1×10-4.With the IC50 of 573.75 ng/mL,No.1 mouse polyclonal antiserum showed the best sensitivity.In this study,AMO-BSA and AMO-OVA were successfully synthesized and high sensitive polyclonal antiserums against AMO were prepared,which provides a basis for the preparation of monoclonal antibodies against AMO.