环丙氨嗪完全抗原的合成及免疫原性鉴定

Synthesis and immunogenicity identification of the cyromazine complete antigen

目的合成并鉴定环丙氨嗪(cyromazine,CA)人工抗原。方法采用碳二亚胺法(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,EDC)将环丙氨嗪与牛血清蛋白(bovine serum albumin,BSA)和鸡卵清蛋白(ovalbumin,OVA)分别偶联;采用紫外分光光度法(ultraviolet spectrophotometry,UV)、聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)和动物免疫对人工抗原进行鉴定。经过4次免疫,间接ELISA测定小鼠血清抗体效价,间接竞争ELISA测定血清灵敏性。结果 UV法和SDS-PAGE法结果初步显示半抗原环丙氨嗪和载体蛋白偶联成功;小鼠血清效价均在1:6400以上,2号小鼠多抗血清半数抑制浓度IC_(50)为27.5 ng/mL。结论本研究成功合成了环丙氨嗪的人工抗原,为环丙氨嗪单克隆抗体制备奠定基础。

Objective To synthesize and identify the artificial antigen of cyromazine. Methods Cyromazine （CA） was coupled with bovine serum albumin （BSA） and ovalbumin （OVA） by using 1-（3-dimethylaminopropyl）- 3-ethylcarbodiimide hydrochloride （EDC） method separately. The artificial antigen was identified by ultraviolet spectrophotometry （UV） scanning, sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and animal immunity. After 4 times of immunization, the titers of antiserum was determined by indirect ELISA, and the sensitivity of antiserum was assayed by indirect competitive ELISA. Results The results of UV scanning and SDS-PAGE showed that CA was successful linked to carrier proteins. The titer of the antiserum of mice were all above 1：6400, the IC5o value of No.2 mouse antiserum was 27.5 ng/mL, conclusion This study successfully synthesizes the cyromazine artificial antigens, which lays the foundation for the preparation of CA monoclonal antibody.