Simultaneous aflatoxin (AFB1) and zearalenone (ZEN) contamination in agro-products have become widespread globally and have a toxic superposition effect. In the present study, we describe a highly sensitive and specific dual lateral flow immunochromatographic assay (dual test strip) for rapid and simultaneous detection of AFB1 and ZEN in food and feed samples based on respective monoclonal antibodies (mAbs). Two immunogens AFB1-BSA (an AFB1 and bovine serum albumin (BSA) conjugate) and ZEN-BSA (a ZEN and BSA conjugate) were synthesized in oximation active ester (OAE) and amino glutaraldehyde (AGA). The molecular binding ratio of AFB1:BSA was 8.64:1, and that of ZEN:BSA was 17.2:1, identified by high-resolution mass spectrometry (HRMS) and an ultraviolet spectrometer (UV). The hybridoma cell lines 2A11, 2F6, and 3G2 for AFB1 and 2B6, 4D9 for ZEN were filtered by an indirect non-competitive enzyme-linked immunosorbent assay (inELISA) and an indirect competitive enzyme-linked immunosorbent assay (icELISA), respectively. As AFB1 mAb 2A11 and ZEN mAb 2B6 had the lowest 50% inhibitive concentration (IC50) and cross-reactivity (CR), they were selected for subsequent experiments. By systematically optimizing the preparation condition of gold nanoparticles (AuNPs), AuNPs-labeled mAbs, and detection condition, the visual limit of detection (LOD) of the dual test strip was 1.0 μg/L for AFB1 and 5.0 μg/L for ZEN, whereas that of the test strip reader was 0.23 μg/L for AFB1 and 1.53 μg/L for ZEN. The high reproducibility and stability of the dual test were verified using mycotoxin-spiked samples. The dual test strips were highly specific and sensitive for AFB1 and ZEN, which were validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Thus, the proposed AFB1 and ZEN dual test strip is suitable for rapid and simultaneous detection of AFB1 and ZEN contamination in food and feed samples.

In this study, we irradiated Astragalus polysaccharides (APS) using 25 kGy ⁶⁰Co γ ray to obtain γ‐irradiated Astragalus polysaccharides (IAPS) and then investigated the effects of IAPS on growth performance and immune function of cyclophosphamide (CPM)‐treated broilers. The physicochemical properties of APS and IAPS (molecular weight, water solubility, viscosity, morphological and structural properties) were evaluated. Then, 384 one‐day‐old Arbor Acres broiler chicks with similar initial weight were randomly assigned into 6 groups: the non‐treated group (control), and CPM‐treated groups were fed either a basal diet or the diets containing 900 mg/kg APS, or 900, 600, 300 mg/kg IAPS, respectively. On days 16, 18, and 20, all broilers except for the control group were intramuscularly injected with 0.5 ml CPM (40 mg/kg·BW). Broilers in the control group were intramuscularly injected with 0.5 ml sterilized saline (0.75%, wt/vol). This trial lasted for 21 days. The physicochemical treatment showed that γ irradiation could decrease the molecular weight and viscosity, and increase the water solubility of APS (p < 0.05), whereas the structural properties of APS was not affected. In the animal trial, 900 mg/kg APS or 900, 600 mg/kg IAPS relieved the decreased growth performance, thymus index, T lymphocytes proliferation, serum IgG concentration, NOS activity and the increased blood heterophil:lymphocyte ratio in CPM‐treated broilers (p < 0.05). CPM‐induced decreases in B lymphocytes proliferation and serum IgM concentration were only increased by IAPS at 900 mg/kg (p < 0.05). Overall, both APS and IAPS alleviated CPM‐induced immunosuppression. Especially, IAPS possessed better immunomodulatory effect than APS, indicating that γ irradiation could be used as an effective method to enhance the immunomodulatory activity of APS.

Zearalenone (ZEN) contamination in food and feed is prevalent and has severe effects on humans and animals post-consumption. Therefore, a sensitive, specific, rapid, and reliable method for detecting a single residue of ZEN is necessary. This study aimed to establish a highly sensitive and specific ZEN monoclonal antibody (mAb) and an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of ZEN residues in food and feed. The immunogen ZEN-BSA was synthesized via the amino glutaraldehyde (AGA) and amino diazotization (AD) methods and identified using 1H nuclear magnetic resonance (1H NMR), a high-resolution mass spectrometer (HRMS), and an ultraviolet spectrometer (UV). The coating antigens ZEN-OVA were synthesized via the oxime active ester (OAE), formaldehyde (FA), 1,4-butanediol diglycidyl ether (BDE), AGA, and AD methods. These methods were used to screen the best antibody/antigen combination of a heterologous icELISA. Balb/c mice were immunized with a low ZEN-BSA dose at long intervals and multiple sites. Suitable cell fusion mice and positive hybridoma cell lines were screened using a homologous indirect non-competitive ELISA (inELISA) and an icELISA. The ZEN mAbs were prepared by inducing ascites in vivo. The immunological characteristics of ZEN mAbs were then assessed. The standard curves of the icELISA for ZEN were constructed under optimal experimental conditions, and the performance of the icELISA was validated. The two ZEN-BSA immunogens (conjugation ratios, 11.6:1 (AGA) and 9.2:1 (AD)) were successfully synthesized. Four hybridoma cell lines (2B6, 4D9, 1A10, and 4G8) were filtered, of which 2B6 had the best sensitivity and specificity. The mAb 2B6-based icELISA was then developed. The limit of detection (LOD), the 50% inhibitive concentration (IC50), and the linear working range (IC20 to IC80) values of the icELISA were 0.76 μg/L, 8.69 μg/L, and 0.92–82.24 μg/L, respectively. The cross-reactivity (CR) of the icELISA with the other five analogs of ZEN was below 5%. Three samples were spiked with different concentrations of ZEN and detected using the icELISA. The average intra-assay recoveries, inter-assay recoveries, intra-assay coefficients of variations (CVs), and inter-assay CVs were 93.48–99.48%, 94.18–96.13%, 12.55–12.98%, and 12.53–13.58%, respectively. The icELISA was used to detect ZEN in various samples. The results were confirmed using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) (correlation coefficient, 0.984). The proposed icELISA was highly sensitive, specific, rapid, and reliable for the detection of ZEN in food and feed samples.

This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 10² in supernatants and (1.28 to 5.12) × 10⁵ in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 μg/L in the supernatants and 18.12 to 31.46 μg/L in the ascites. The affinity constant (Ka) of all of the mAbs was between 4.15 × 10⁹ and 6.54 × 10⁹ L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 μg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 μg/L, and its linear working range was between 1.03 and 288.55 μg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed.

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.