Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics

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 11.67 μg/L (OAE), 16.29 μg/L (CMA), 20.92 μg/L (FA) and 24.36 μg/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.

Development of a Highly Sensitive and Specific Monoclonal Antibody Based on Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Zearalenone in Food and Feed Samples

“…Spacer arms that are too long or too short are not suitable for the full exposure of the characteristic structure of the hapten, thereby affecting the recognition by immune cells [ 29 ]. As a result, the C5 position of the benzene ring of the ZEN molecule was selected as the active site for the synthesis of 5-NH 2 -ZEN as the hapten via nitration and reduction reaction based on the comparison and analysis of the ZEN hapten’s molecular design, the immunogen synthesis, and the characteristics of the prepared specific antibodies reported in the previous literature ( Table 1 ) and in our previous research reports [ 30 ]. The AGA and AD methods were used to synthesize two new immunogens by introducing a five-carbon chain structure and an unsaturated N=N structure as spacer arm, respectively.…”

“…The buffers and solutions used in this study were the same as those used previously [ 30 ]. Phosphate buffer saline (PBS; 0.01 M, pH 7.4) contained 137 mM NaCl, 10 mM Na 2 HPO 4 ·12H 2 O, 2.68 mM KCl, and 1.47 mM KH 2 PO 4 .…”

Indirect Competitive ELISA for the Determination of Total Chromium Content in Food, Feed and Environmental Samples

“…The cryopreserved hybridoma cells were resuscitated and passaged once every ten days for five times. Both inELISA and icELISA were used to detect the antibody titers and IC50 values of Cr(III)-EDTA in the supernatants at different passages to determine the stability of the hybridoma cells secreting an antibody [ 46 ].…”

Alternative Hapten Design for Zearalenone Immunoreagent Generation

“…α-ZAL is used as a growth promoter in some countries under the name of zeranol; however, this compound is banned in the EU, hence it is included in many official control programs [10]. The approaches so far reported for synthesizing ZEN haptens have mostly involved the use of a simple ZEN-oxime derivative at position C-7 [11,12]. In our opinion, this chemical strategy suggests relevant alterations in the stereoelectronic properties and conformation of the parent molecule.…”

“…The approaches so far reported for synthesizing ZEN haptens have mostly involved the use of a simple ZEN-oxime derivative at position C-7 [ 11 , 12 ]. In our opinion, this chemical strategy suggests relevant alterations in the stereoelectronic properties and conformation of the parent molecule.…”