Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics

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Abstract
Background. This study aimed to explore the zearalenone (ZEN) immunogen synthesis method, immunogenicity, and antibody characteristics and to lay a foundation for the establishment of immunoassay methods for ZEN single residue and ZEN and its analogs total residue. Methods. Based on the molecular structure and active sites of ZEN, oxime active ester (OAE), condensation mixed anhydride (CMA), formaldehyde (FA), and 1,4-butanediol diglycidyl ether method (BDE) were designed and used for immunogen (ZEN-BSA) synthesis. The immunogens were identified by infrared (IR) and ultraviolet (UV) spectra and gel electrophoresis (SDS-PAGE) and were then used to immunize Balb/c mice to prepare ZEN polyclonal antibody (ZEN pAb). The titers and sensitivity of the ZEN pAb were determined by indirect noncompetitive ELISA (inELISA) and indirect competitive ELISA (icELISA), respectively, and its specificity was assessed by the cross-reaction test (CR). Results. ZEN-BSA was successfully synthesized, and the molecular binding ratios of ZEN to BSA were 17.2:1 (OAE), 14.6:1 (CMA), 9.7:1 (FA), and 8.3:1 (BDE), respectively. The highest inELISA titers of ZEN pAb of each group were 1:(6.4×103) (OAE), 1:(3.2×103) (CMA), 1:(1.6×103) (FA), and 1:(1.6×103) (BDE), respectively. The 50 inhibition concentrations (IC50) for ZEN by icELISA of each group were 11.67μg/L (OAE), 16.29μg/L (CMA), 20.92μg/L (FA) and 24.36μg/L (BDE), respectively. ZEN pAb from the mice immunized with ZEN-BSA (OAE) and ZEN-BSA (CMA) had class broad specificity to ZEN and its analogs. The CRs of ZEN pAb with α-ZAL, β-ZAL, α-ZOL, β-ZOL, and ZON were 36.53, 16.98, 64.33, 20.16, and 10.66, respectively. ZEN pAb from the mice immunized with ZEN-BSA (FA) and ZEN-BSA (BDE) had high specificity for ZEN. The CRs of ZEN pAb with its analogs were all less than 1.0. Conclusion. This study demonstrated that the preparation of the class broad-specificity antibodies of ZEN and its analogs can be achieved by immunizing animals with the immunogen ZEN-BSA prepared by the OAE method, while the preparation of highly specific antibodies can be achieved by immunizing animals with the immunogen ZEN-BSA prepared by the FA method. These findings lay the material and technical foundation for immunoassay of ZEN single residue and ZEN and its analogs total residue.
Funding Information
  • National Natural Science Foundation of China (31702263, 2019YFC1605705, 20IRTSTHN025)
  • 13th Five-Year National Key Research and development plan food safety technology research and development major project (31702263, 2019YFC1605705, 20IRTSTHN025)
  • Henan University (31702263, 2019YFC1605705, 20IRTSTHN025)

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