Synthesis of Ethyl Vanillin Artificial Antigen and Preparation of Murine Polyclonal Antibodies
In order to study the immunological methods for detecting ethyl vanillin(EV),ethyl vanillic acid(EVA)was coupled with bovine serum albumin(BSA)by the active ester(AE)and mixed anhydride(MA)methods to prepare the immune antigens BSA-EV(AE)and BSA-EV(MA).UV scanning and SDS-PAGE were used to identify the artificial antigens.BALB/c mice were immunized with BSA-EV(AE)or BSA-EV(MA),tail-cut blood samples were collected and serum were separated at 10 days after the third immunization to prepare rat polyclonal antibodies.Serum titer was determined by indirect ELISA,and serum sensitivity and specificity were determined by indirect competitive ELISA.The results showed that the artificial antigen of EV was prepared successfully,and the coupling ratios of BSA-EV(AE)and BSA-EV(MA)were 1∶2.92 and 1∶37.95.The serum titer of the No.3 mouse of BSA-EV(AE)immunization group was the highest with 1∶51 200,the serum half inhibitory concentration(IC50)against EV was 173.78 μg/L,the liner range was 14.79-2 041.74 μg/L.The serum titer of the No.1 mouse of BSA-EV(MA)immunization group was the highest with 1∶102 400,the serum half inhibitory concentration(IC50)against EV was 617.00 μg/L,the liner range was 42.66-8 709.64 μg/L.The cross reaction rate of serum antibodies in the two immunization groups with vanillin,methyl vanillin,maltol,ethyl maltol,BSA,ovalbumin(OVA)was less than 1%.In conclusion,the artificial antigens of EV were successfully synthesized,and mouse polyclonal antibodies against EV with high sensitivity and specificity were prepared by animal immunization.
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