Synthesis of Zearalenone Immunogen and Comparative Analysis of Antibody Characteristics.

Yanan Wang,Haitang Zhang,Xiaofei Wang,Jinqing Jiang,Hanna Fotina,Adil Denizli

doi:10.1155/2021/7109383

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.

Highlights

Zearalenone (ZEN) is a toxic secondary metabolite produced by members of the genus Fusarium, and ZEN mainly contaminates grains including corn, wheat, barley, rice, and oats or foods containing these grains, and its chemical name is (E)-(S)-2,4-dihydroxy-7-methyl-7,8,9,10,13,14-hexahydro-12h-6-oxa-benzocyclotetradecene-5,11-dine, called F-2 toxin. e ZEN analogs produced under natural conditions mainly include α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), and zearalanone (ZON) (Figure 1) [1, 2]
Comparing the IR of the artificial immunogen ZEN-Bovine serum albumin (BSA) synthesized via the four methods with BSA, we revealed similar IR absorption in the regions 2800–3200 cm−1 and 1100–1700 cm−1, which were the characteristic peaks produced by the amine group and amide group in BSA (Figure 6)
BSA had no absorption in the same areas. e characteristic peaks produced by hydroxyl and esters in ZEN indicated that the artificial immunogen ZEN-BSA synthesized by the four methods contain ZEN. ese findings revealed that the four methods successfully synthesized the artificial immunogen ZEN-BSA

Summary

Introduction

Zearalenone (ZEN) is a toxic secondary metabolite produced by members of the genus Fusarium, and ZEN mainly contaminates grains including corn, wheat, barley, rice, and oats or foods containing these grains, and its chemical name is (E)-(S)-2,4-dihydroxy-7-methyl-7,8,9,10,13,14-hexahydro-12h-6-oxa-benzocyclotetradecene-5,11-dine, called F-2 toxin. e ZEN analogs produced under natural conditions mainly include α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), and zearalanone (ZON) (Figure 1) [1, 2]. Zearalenone (ZEN) is a toxic secondary metabolite produced by members of the genus Fusarium, and ZEN mainly contaminates grains including corn, wheat, barley, rice, and oats or foods containing these grains, and its chemical name is (E)-(S)-2,4-dihydroxy-7-methyl-7,8,9,10,13,14-hexahydro-12h-6-oxa-benzocyclotetradecene-5,11-dine, called F-2 toxin. Owing to the reproductive toxicity, genotoxicity, immunotoxicity, endocrine toxicity, and carcinogenic toxicity of ZEN to the body [3, 4], most countries and regions globally have implemented the maximum residue limits (MRLs) of ZEN in food and feed. E EU stipulates that the MRL of ZEN in cereals and grain products is 2 mg/kg; the MRL of ZEN in corn by-products is 3 mg/kg; the MRL of ZEN in compound feeds for piglets and young sows are 0.1 mg/kg [5].

Methods

Results

Discussion

Conclusion