Na Liu

Multiplex Lateral Flow Immunoassay for Mycotoxin Determination

A new lateral flow immunoassay (LFA) is proposed for qualitative and/or semiquantitative determination of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON), and their analogues (AFs, ZEAs, DONs) in cereal samples. Each of the mycotoxin specific antibody was class specific and there was no cross reactivity to other groups of compounds. The visual limits of detection (vLOD) of the strip were 0.03, 1.6, and 10 μg/kg for AFB1, ZEA and DON, respectively. The calculated limits of detection (cLOD) were 0.05, 1, and 3 μg/kg, respectively. Meanwhile the cutoff values were achieved at 1, 50, and 60 μg/kg for AFB1, ZEA and DON, respectively. Recoveries ranged from 80% to 122% and RSD from 5% to 20%. Both the vLOD and cLOD for the three mycotoxins were lower than the EU maximum levels. Analysis of naturally contaminated maize samples resulted in a good agreement between the multiplex LFA and LC-MS/MS (100% for DONs and AFs, and 81% for ZEAs). Careful analysis of the results further explained the general overestimation of LFA compared to chromatographic methods for quantification of mycotoxins.

Functional Agents to Biologically Control Deoxynivalenol Contamination in Cereal Grains.

Mycotoxins, as microbial secondary metabolites, frequently contaminate cereal grains and pose a serious threat to human and animal health around the globe. Deoxynivalenol (DON), a commonly detected Fusarium mycotoxin, has drawn utmost attention due to high exposure levels and contamination frequency in the food chain. Biological control is emerging as a promising technology for the management of DON contamination. Functional biological control agents (BCAs), which include antagonistic microbes, natural fungicides derived from plants and detoxification enzymes, can be used to control DON contamination at different stages of grain production. In this review, studies regarding different biological agents for DON control in recent years are summarized for the first time. Furthermore, this article highlights the significance of BCAs for controlling DON contamination, as well as the need for more practical and efficient BCAs concerning food safety.

Simultaneous determination of major type B trichothecenes and deoxynivalenol-3-glucoside in animal feed and raw materials using improved DSPE combined with LC-MS/MS.

A simple and reliable method for simultaneous determination of deoxynivalenol-3-glucoside and major type B trichothecenes (deoxynivalenol, nivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol and deepoxy-deoxynivalenol) in animal feed and raw materials has been developed and validated in this study. The method was based on an improved dispersive solid-phase extraction (DSPE) followed by analysis using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Also, matrix-matched calibration curve (R(2)>0.99) was employed to minimize matrix effects and ensure accurate quantification. The recoveries during sample preparation process (including extraction and clean-up) ranged from 79.03% to 118.39%, with intra-day and inter-day relative standard deviation lower than 20% for all the analytes. The limit of quantification ranged from 5.0 μg/kg for deoxynivalenol to 13.6 μg/kg for fusarenon X. The validated method was successfully applied to the analysis of animal feed and corn. The pilot study showed that 37 out of 41 samples were contaminated with deoxynivalenol-3-glucoside at the levels of 6.0-121.0 μg/kg. Most of the type B trichothecenes were also found with the exception of fusarenon X, at the contaminated levels of 10.0-1,382 μg/kg. To the best of our knowledge, this was the first scientific report on the co-occurrence of masked deoxynivalenol and type B trichothecenes in animal feed and raw materials.

Multi-mycotoxin analysis of animal feed and animal-derived food using LC–MS/MS system with timed and highly selective reaction monitoring

AbstractMycotoxins have the potential to enter the human food chain through carry-over of contaminants from feed into animal-derived products. The objective of the study was to develop a reliable and sensitive method for the analysis of 30 mycotoxins in animal feed and animal-derived food (meat, edible animal tissues, and milk) using liquid chromatography–tandem mass spectrometry (LC–MS/MS). In the study, three extraction procedures, as well as various cleanup procedures, were evaluated to select the most suitable sample preparation procedure for different sample matrices. In addition, timed and highly selective reaction monitoring on LC–MS/MS was used to filter out isobaric matrix interferences. The performance characteristics (linearity, sensitivity, recovery, precision, and specificity) of the method were determined according to Commission Decision 2002/657/EC and 401/2006/EC. The established method was successfully applied to screening of mycotoxins in animal feed and animal-derived food. The results indicated that mycotoxin contamination in feed directly influenced the presence of mycotoxin in animal-derived food. Graphical abstractMulti-mycotoxin analysis of animal feed and animal-derived food using LC-MS/MS

A rabbit monoclonal antibody-based sensitive competitive indirect enzyme-linked immunoassay for rapid detection of chloramphenicol residue

A sensitive competitive indirect enzyme-linked immunoassay (ciELISA) based on a rabbit monoclonal antibody (RabMAb) against chloramphenicol (CAP) has been developed and validated in this study. After optimisation of several key physicochemical factors, such as Tween-20 percentage, pH value and ionic strength, the immunoassay showed excellent performance within the linear range of 0.18–6.37 ng mL−1, with the 50% inhibition concentration (IC50) of 1.06 ng mL−1 and the limit of detection (LOD) of 0.1 ng mL−1. In addition, the cross-reactivities of RabMAb towards chloramphenicol succinate, florfenicol and thiamphenicol were 2.09, 12.45 and 18.10%, respectively. Finally, the developed method was applied in spiked swine urine, milk and honey samples, with recoveries ranging from 71.03 to 109.62%. The result demonstrated that the developed immunoassay is a valuable method for screening and quantitation of CAP residues in real samples.

Detoxification of Deoxynivalenol via Glycosylation Represents Novel Insights on Antagonistic Activities of Trichoderma when Confronted with Fusarium graminearum

Deoxynivalenol (DON) is a mycotoxin mainly produced by the Fusarium graminearum complex, which are important phytopathogens that can infect crops and lead to a serious disease called Fusarium head blight (FHB). As the most common B type trichothecene mycotoxin, DON has toxic effects on animals and humans, which poses a risk to food security. Thus, efforts have been devoted to control DON contamination in different ways. Management of DON production by Trichoderma strains as a biological control-based strategy has drawn great attention recently. In our study, eight selected Trichoderma strains were evaluated for their antagonistic activities on F. graminearum by dual culture on potato dextrose agar (PDA) medium. As potential antagonists, Trichoderma strains showed prominent inhibitory effects on mycelial growth and mycotoxin production of F. graminearum. In addition, the modified mycotoxin deoxynivalenol-3-glucoside (D3G), which was once regarded as a detoxification product of DON in plant defense, was detected when Trichoderma were confronted with F. graminearum. The occurrence of D3G in F. graminearum and Trichoderma interaction was reported for the first time, and these findings provide evidence that Trichoderma strains possess a self-protection mechanism as plants to detoxify DON into D3G when competing with F. graminearum.

Development of a new rabbit monoclonal antibody and its based competitive indirect enzyme-linked immunosorbent assay for rapid detection of sulfonamides.

BACKGROUND Monoclonal antibodies generally obtained through the classic mouse hybridoma system were requisite for the establishment of various immunoassays. In this study, a new rabbit monoclonal antibody (RabMAb) against sulfonamides (SAs) was first produced via hybridoma technique in rabbit. The related enzyme-linked immunosorbent assay (ELISA) was then developed and applied to real sample analysis. RESULTS A sensitive competitive indirect ELISA method based on a novel RabMAb for rapid detection of sulfonamides was first established. The obtained half-maximum inhibition concentration (IC(50)) values for four SAs were all below 10 ng mL(-1) , with 0.68 ng mL(-1) sulfathiazole (STZ), 1.11 ng mL(-1) sulfadiazine (SD), 1.15 ng mL(-1) sulfapyridine (SP) and 5.27 ng mL(-1) sulfamethoxazole (SMX). Desirable recoveries when detecting different spiked swine urine and milk samples were achieved ranging from 92.6% to 104.3% and from 61.1% to 81.6%, respectively. CONCLUSION The proposed immunoassay with the newly developed RabMAb is capable of detection of four SAs (STZ, SD, SP and SMX) with proven satisfactory performance and is applicable for routine large-scale analysis in practical uses.

Simultaneous Raising of Rabbit Monoclonal Antibodies to Fluoroquinolones with Diverse Recognition Functionalities via Single Mixture Immunization

Highly specific monoclonal and polyclonal antibodies are the key components in a diverse set of immunoassay applications, from research work to routine monitoring and analysis. In the current manuscript, combinatorial strategies for a single mixture immunization, screening and rabbit hybridoma cell technology were described. Fluoroquinolones (FQs) drugs were chosen as representative analytes. Six FQs were conjugated with bovine serum albumin and used as immunogens for subsequent immunization, while a mixture of all was injected for coimmunization. The hybridomas obtained against the individual and multiple FQs were used for the production of diverse varieties of rabbit monoclonal antibodies (RabMAbs) against the target analytes. As was proven by indirect competitive ELISA and quantitative lateral flow immunoassay, this approach opens a new way for simultaneously obtaining functional monoclonal antibodies which are capable of recognizing both individual and multiple analytes in a single preparation circle. This addresses various needs of different monitoring regulations as analytical methodology advances.

Individual and Combined Cytotoxic Effects of Co‐Occurring Deoxynivalenol Family Mycotoxins on Human Gastric Epithelial Cells

Mycotoxin contamination is a significant health concern for human beings, but health risk assessments are usually based on one single mycotoxin, which might neglect the additive or competitive interactions between co-occurring mycotoxins. In this study, we assessed the individual or combined toxicological effects to multiple deoxynivalenol-family mycotoxins, namely deoxynivalenol (DON), Nivalenol (NIV), and their acetyl derivatives of 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), deoxynivalenol-3-glucoside (D3G), and Fusarenon-X (FX) based on the human gastric epithelial (GES-1) cells. GES-1 cells were treated at different concentrations over 24 h and cell viability was measured by a cell counting kit (CCK8). The results show that D3G has no toxicity and 3-ADON is less potent in reducing cell viability compared to DON, whereas 15-ADON and FX appear to be slightly less potent than their parent compounds of DON and NIV on GES-1 cells. In general, the toxic ability of individual mycotoxins was shown as 3-ADON << 15-ADON < DON < FX < NIV, in an increasing order. All mixtures caused a dose-dependent decline of cell viability and the interactions analysis of binary combinations were assessed using the combination index (CI)-isobologram method. For the interaction types of mycotoxins mixtures, the synergistic cytotoxicity of DON + 15-ADON, DON + NIV, and DON + FX at low and/or moderate inhibitory concentration levels (IC10–IC70, IC10–IC80, and IC10–IC40, respectively) were observed. FX + NIV resulted in almost completely synergistic cytotoxicity, whereas 15-ADON + NIV and 15-ADON + FX presented almost entirely antagonistic cytotoxicity on the GES-1 cell model. These results suggest that the simultaneous presence of low-dose type B trichothecenes in dietary food may be more or less toxic than the prediction based on individual mycotoxins.

Large-scale preparation and multi-dimensional characterization of high-purity mycotoxin deoxynivalenol in rice culture inoculated with Fusarium graminearum

Food safety monitoring and toxicological research of mycotoxins are still in need of large quantities of high-purity deoxynivalenol (DON). To attain this purpose, a rapid, economical and reproducible purification method was developed for large-scale production of DON from rice culture inoculated with a DON-producing Fusarium graminearum strain JYH. The inoculated rice culture was first extracted with acetonitrile–water (84/16, v/v). The extracts were evaporated to dryness on a rotary evaporator after ethyl acetate partitioning and then dissolved in water followed by the final purification procedure through preparative high performance liquid chromatography and montmorillonite treatment. A combined approach of ultraviolet spectrometry (UV), ultra high performance liquid chromatography (UHPLC), mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy was applied for the multi-dimensional characterization of the target compound. As a result, the recovery of DON from the crude extract to the final product was up to 70%. An amount of 150 mg DON with the desirable purity of 98.93% could be obtained from 100 g of rice culture, which possessed identical immunochemical characteristics compared to a certified commercial DON standard. This proposed strategy might act as a valuable reference to obtain rather expensive compounds in a straightforward way.