玉米赤霉醇完全抗原的制备及质量鉴定
Preparation and Quality Identification of Complete Antigen of Zeranol

旨在合成玉米赤霉醇（ZER）人工抗原，并用其免疫小鼠以获得含高滴度ZER多克隆抗体的小鼠血清，为ZER单克隆抗体的制备奠定基础。改造ZER第16位上的羟基，合成半抗原ZER-16-羧丙基丁醚，然后分别采用混合酸酐法和碳二亚胺（EDC）法将改造后的半抗原与载体蛋白BSA或OVA偶联，制备免疫原ZER-BSA和包被原ZER-OVA，并采用凝胶电泳、动物免疫对人工抗原进行质量鉴定，间接ELISA测定抗血清效价，间接竞争（阻断）ELISA测定抗血清的敏感性和特异性。结果表明，用制备的免疫原免疫小鼠血清效价均已达到1∶10 以上。6号鼠的血清敏感性最高，对ZER的半数抑制质量浓度（IC50）达15.77 ng/mL，获得的血清除与 α-玉米赤霉醇特异性反应外，与其结构类似物β-玉米赤霉醇、 α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮、玉米赤霉烯酮分别有12.5%、100%、12.5%、25%、100%的交叉反应，与其他霉菌毒素交叉反应性均小于0.5%。表明试验成功获得ZER人工抗原，通过动物免疫制备敏感性好、特异性强的ZER多克隆抗体，为ZER单克隆抗体的制备及其免疫学检测方法的建立奠定基础。
The purpose of this study is to synthesize complete antigen of ZERanol (ZER),and to get the mice that produce high sensitivity antiserum against ZER by immunizing mice with the artificial antigen,which will lay the foundation for preparation of ZER monoclonal antibody.ZER-(16-hydroxypropyl butyl ether ) was synthesized by modifying the sixteenth hydroxyl of ZER.The complete antigen ZER-BSA and ZER-OVA were obtained by coupling ZER-(16-hydroxypropyl butyl ether ) with carrier proteins BSA and OVA using mixed anhydrides method and Carbodiimide method,respectively.The quality of complete antigen was identified by SDS-PAGE and animal immunity.The titer of polyclonal antiserum was detected by indirect ELISA,and sensitivity and specificity by indirect competition (block) ELISA.The results showed that the antiserum titer of the immunized mice were more than 1∶10. The antiserum sensitivity of No.6 mouse was the best among the immunized mice,which median inhibitory mass concentration (IC50) against ZER was 15.77 ng/mL,the antiserum specific reacts with ZER.Moreover,the cross-reaction of antiserum toward similar structure such as β-ZER,α-zearalenol,β-zearalenol,zearalanone,zearalenone were 12.5%,100%,12.5%,25%,100%,respectively,and toward other mycotoxins or carrier proteins were all less than 0.5%.In conclusion,we synthesized complete antigen of ZERanol successfully,and obtained high sensitivity,specificity antiserum,which laid the foundation for the preparation of monoclonal antibody and the establishment of immunological detection technique of ZER.