Wang Yanan, Wang Xiaofei, Zhang Haitang, Jiang Jinqing, Fotina Hanna
College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, China.
Faculty of Veterinary Medicine, Sumy National Agrarian University, Sumy 40021, Ukraine.
Int J Anal Chem. 2021 Jul 26;2021:7109383. doi: 10.1155/2021/7109383. eCollection 2021.
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.
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).
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 × 10) (OAE), 1 : (3.2 × 10) (CMA), 1 : (1.6 × 10) (FA), and 1 : (1.6 × 10) (BDE), respectively. The 50% inhibition concentrations (IC50) for ZEN by icELISA of each group were 11.67 g/L (OAE), 16.29 g/L (CMA), 20.92 g/L (FA) and 24.36 g/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 -ZAL, -ZAL, -ZOL, -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%.
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.
本研究旨在探索玉米赤霉烯酮（ZEN）免疫原的合成方法、免疫原性及抗体特性，为建立ZEN单残留及ZEN及其类似物总残留的免疫分析方法奠定基础。
基于ZEN的分子结构和活性位点，设计并采用肟活性酯（OAE）法、混合酸酐缩合法（CMA）、甲醛（FA）法和1,4 - 丁二醇二缩水甘油醚法（BDE）合成免疫原（ZEN - BSA）。通过红外光谱（IR）、紫外光谱（UV）和凝胶电泳（SDS - PAGE）对免疫原进行鉴定，然后用其免疫Balb/c小鼠制备ZEN多克隆抗体（ZEN pAb）。分别采用间接非竞争性ELISA（inELISA）和间接竞争性ELISA（icELISA）测定ZEN pAb的效价和灵敏度，并通过交叉反应试验（CR）评估其特异性。
成功合成了ZEN - BSA，ZEN与BSA的分子结合比分别为17.2∶1（OAE法）、14.6∶1（CMA法）、9.7∶1（FA法）和8.3∶1（BDE法）。各组ZEN pAb的最高inELISA效价分别为1∶(6.4×10)（OAE法）、1∶(3.2×10)（CMA法）、1∶(1.6×10)（FA法）和1∶(1.6×10)（BDE法）。各组icELISA对ZEN的50%抑制浓度（IC50）分别为11.67μg/L（OAE法）、16.29μg/L（CMA法）、20.92μg/L（FA法）和24.36μg/L（BDE法）。用ZEN - BSA（OAE）和ZEN - BSA（CMA）免疫的小鼠所产生的ZEN pAb对ZEN及其类似物具有较宽的类特异性。ZEN pAb与α - ZAL、β - ZAL、α - ZOL、β - ZOL和ZON的交叉反应率分别为36.53%、16.98%、64.33%、20.16%和10.66%。用ZEN - BSA（FA）和ZEN - BSA（BDE）免疫的小鼠所产生的ZEN pAb对ZEN具有高特异性。ZEN pAb与其类似物的交叉反应率均小于1.0%。
本研究表明，用OAE法制备的免疫原ZEN - BSA免疫动物可制备ZEN及其类似物的类宽特异性抗体，而用FA法制备的免疫原ZEN - BSA免疫动物可制备高特异性抗体。这些研究结果为ZEN单残留及ZEN及其类似物总残留的免疫分析奠定了物质和技术基础。
