| Abstract: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. |
