TY - CONF A1 - Vergin, Chantal C. T1 - Development of antibodies against mycotoxins and their application in immunochemical methods N2 - Food safety is a central and topical issue in our society and is governed by food law. Mycotoxins are secondary metabolic products formed by molds. These contaminants can enter food and thus the food chain through infestation, posing a serious health risk to humans and animals. For this reason, the European Commission has issued Regulation (EU) 2023/915, which sets maximum levels for certain mycotoxins in food. Currently, around 25 % of foodstuffs are contaminated with mycotoxins above the legally prescribed limits. Regular checks are essential to prevent such exceedances. The analytical methods currently available, mainly based on chromatographic techniques such as LC-MS/MS, are considered inadequate for on-site use – i.e., at processing and production facilities in the food industry – because they are technically complex and labor-intensive. One possible improvement is the use of immunoassays, which are widely accepted and employed in medical diagnostics. However, a basic prerequisite for developing such assays is the availability of specific antibodies. Our focus in this project is the development of high-affinity, highly selective monoclonal antibodies against mycotoxins for which either no antibodies, only polyclonal antibodies, or antibodies with insufficient specificity are currently available. The goal is to generate antibodies targeting patulin, Alternaria toxins, and ergot alkaloids for use in rapid tests and on-site analytical systems. A key aspect is the group selectivity of the antibodies with regard to the various ergot alkaloids. Depending on the mycotoxin, heterologous or homologous haptens are used to immunize mice. To ensure monoclonality, we apply limiting dilution and antigen-specific fluorescence-activated cell sorting (FACS) during the selection process. The antibodies developed will be used to expand SAFIA Technologies GmbH’s existing mycotoxin test-kit, enabling reliable detection of mycotoxins in food and thereby contributing to improved food safety standards. We will showcase our approach along with the results achieved so far T2 - 5th European Biosensor Symposium CY - Tarragona, Spain DA - 26.10.2025 KW - Antibodies KW - Biosensor KW - Immunoassay KW - Mycotoxins PY - 2025 AN - OPUS4-64459 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Herter, Sven-Oliver T1 - Quantifying ergot alkaloids in food using stable isotopically labeled standards N2 - Ergot alkaloids (EAs) are toxic secondary metabolites produced by fungi of the genus Claviceps. They grow on rye and wheat, and are introduced into the food chain through the harvest of infected cereals. Therefore, the European Union has established a maximum level for the 12 most abundant EAs. In order to improve the quantification, stable isotopically labelled EAs were synthesized for the first time and their performance was evaluated in comparison to the current European standardmethod in different foodstuff. T2 - ANAKON 2025 CY - Leipzig, Germany DA - 10.03.2025 KW - Reference Material KW - HPLC-MS/MS KW - Mycotoxins KW - Standards KW - Stable Isotope Dilution Analysis PY - 2025 AN - OPUS4-62752 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Herter, Sven-Oliver T1 - Semi-synthesis of isotopic labeled ergot alkaloids: New reference standards N2 - We developed a two-step semi-synthesis for the preparation of isotopically labeled EAs, starting from native EAs. This universal strategy enabled the successful synthesis of all isotopically labeled priority EAs. The structure of the isotopically labeled EAs was confirmed by HPLC-HR-MS/MS using native, unlabeled EAs as a reference standard. The next step will be the implementation of the isotopically labeled standards in the European standard procedure EN 17425 to improve the quantification of EAs in foodstuffs. T2 - 45th Mycotoxin Workshop CY - Vienna, Austria DA - 02.06.2024 KW - Reference Material KW - HPLC-MS/MS KW - Mycotoxins KW - Isotop standards KW - Organic Synthesis KW - Ergot alkaloids PY - 2024 AN - OPUS4-60239 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Herter, Sven-Oliver T1 - Synthesis and application of stable isotopically labeled ergot alkaloids N2 - Ergot alkaloids form a toxicologically relevant group of mold toxins (mycotoxins) that are among the most common contaminants of food and animal feed worldwide. Therefore, reliable controls are indispensable for the minimization of both health risks and economic damages. As a consequence of their toxicological significance, EU limit values for 12 priority ergot alkaloids were established for the first time in 2022. These limits range from 500 µg/kg in rye milling products down to 20 µg/kg in processed cereal-based foods for infants and young children. Despite the use of high-performance liquid chromatography-mass spectrometry to quantify low concentrations of ergots in food, the current European standard procedure EN 17425:2021 cannot be fully applied due to the lack of isotopically labeled reference standards. The complex structure of the ergot alkaloids makes a total synthesis extremely challenging, expensive, and time-consuming. Consequently, we focused on a semi-synthetic approach with the aim of specifically demethylating the N6-atom of the lysergic acid moiety, a shared structural feature among all ergot alkaloids. This resulted in the formation of a norergot alkaloid, which was purified using preparative HPLC. The reaction of the norergot alkaloid with an isotopically labeled electrophilic methyl source, such as iodomethane or dimethyl sulfate, yielded the desired isotopically labeled ergot alkaloid. This methodology enabled the successful synthesis of all 12 stable isotopically labeled priority ergot alkaloids for the first time. Herein we present the problem of the current unavailability of these isotopically labeled standards and our approach to solve this urged demand. Moreover, we are able to present initial data on how these standards enhance the European standard procedure, EN 17425:2021. T2 - 11th International Symposium on RECENT ADVANCES IN FOOD ANALYSIS CY - Prague, Czech Republic DA - 05.11.2024 KW - Reference Material KW - HPLC-MS/MS KW - Mycotoxins KW - Ergot Alkaloids KW - Isotopic Labeling PY - 2024 AN - OPUS4-61690 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Carl, Peter T1 - Progressing Towards Rapid Multiplex Detection: A Fluorescence Immunoassay for Ergot Alkaloids, Trichothecenes, and Fusarium Toxins N2 - Ergot alkaloids, potent mycotoxins produced by Claviceps spp., particularly Claviceps purpurea, pose significant health risks when they contaminate rye and related cereals, leading to ergotism in humans and mammals [1]. In response, the European Union has established Regulation 2023/915, setting maximum residue levels for the sum of 12 principal ergot alkaloids and other mycotoxins including fumonisins (FUM), deoxynivalenol (DON), zearalenone (ZEN), and T2/HT2 toxins in food products. Given the prevalent co-occurrence of mycotoxins, their simultaneous detection is crucial for ensuring the safety of food and feed [2, 3]. Traditionally, chromatographic techniques such as liquid chromatography coupled with (tandem) mass spectrometry (LC-MS/MS) have been employed for multiplex detection of mycotoxins [4, 5]. While effective, these methods require specialized facilities, expensive equipment, and skilled personnel. Immunoassays like ELISA and lateral flow assays offer a more accessible alternative for rapid mycotoxin detection, yet they generally lack the capability for concurrent multi-toxin screening. This study introduces the SAFIA (Suspension Array Fluorescence Immunoassay), a particle-based immunoassay utilizing fluorescence-encoded microparticles for the simultaneous detection of multiple analytes [6, 7]. The assay's innovative advancement comes with the inclusion of ergot alkaloids, a novel addition to its existing detection capabilities for fusarium toxins and trichothecenes, thereby expanding its scope to a broader range of mycotoxins. The assay employs antibodies targeting the ergoline moiety common to all major ergot alkaloids, facilitated by a synthesized hapten mimicking the ergoline structure. This hapten was conjugated to amino-functionalized beads, and a panel of five monoclonal antibodies was evaluated for hapten recognition, binding specificity, and competitive binding efficiency. Our findings demonstrate that all antibodies displayed similar affinities towards the hapten and lysergol (a stable and less hazardous analogue of lysergic acid used for calibration), achieving detection limits as low as 2 ppb. Cross-reactivity studies and analysis of round-robin test material indicated a significant underestimation of ergot alkaloid levels in samples. However, accurate detection of ergot alkaloids remains feasible through the application of a correction factor to the results, which compensates for this underestimation and ensures the assay's effectiveness. Despite this adjustment, the necessity for enhancements in antibody specificity to improve assay accuracy is evident. Furthermore, the inclusion of the ergot assay in a multiplexed setup for detecting FUM, DON, ZEN, and T-2 toxins showed no interference, although an unexpected inhibition among four out of five ergot antibodies was observed. This underscores the need for an improved immunogen structure to achieve optimal detection of ergot alkaloids. In conclusion, our study presents a promising approach for the multiplexed detection of ergot alkaloids alongside other mycotoxins, highlighting the potential of SAFIA in enhancing food and feed safety through improved mycotoxin screening. Future work will focus on refining antibody specificity and assay configurations to overcome current limitations and ensure accurate, comprehensive mycotoxin detection. T2 - Mycotoxin-Workshop CY - Vienna, Austria DA - 02.06.2024 KW - Mycotoxins KW - LC-MS/MS KW - Quality assurance KW - Food safety PY - 2024 AN - OPUS4-61065 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Herter, Sven-Oliver T1 - Isotopically labeled ergot alkaloids: A step toward safer foodstuffs N2 - Ergot alkaloids form a toxicologically relevant group of mold toxins (mycotoxins) that are among the most common contaminants of food and animal feed worldwide. It is therefore imperative that reliable controls are necessary in order to minimize both the health risks and the economic damage. In consequence of their toxicological relevance, EU limit values for 12 priority ergot alkaloids were established for the first time in 2022. These limits range from 500 µg/kg in rye milling products down to 20 µg/kg in processed cereal-based foods for infants and young children. Despite the use of high-performance liquid chromatography-mass spectrometry to quantify low concentrations of ergots in food, the European standard analytical procedure cannot be fully applied due to the unavailability of isotopically labeled reference standards. The complex structure of the ergot alkaloids makes a total synthesis extremely challenging, expensive, and time-consuming. Consequently, we focused on a semi-synthetic approach with the aim of specifically demethylating the N6-atom of the lysergic acid moiety, a shared structural feature among all ergot alkaloids. This resulted in the formation of a norergot alkaloid, which was purified using preparative HPLC. The reaction of the norergot alkaloid with an isotopically labeled electrophilic methyl source, such as iodomethane or dimethyl sulfate, yielded the desired isotopically labeled ergot alkaloid. This methodology enabled the successful synthesis of all 12 stable isotopically labeled priority ergot alkaloids worldwide for the first time. Herein we present the problem of unavailability of these isotopically labeled standards and our approach to solve this urged demand. T2 - Adlershofer Forschungsforum CY - Berlin, Germany DA - 11.11.2024 KW - Reference Material KW - HPLC-MS/MS KW - Mycotoxins KW - Ergot Alkaloids KW - Isotopic Labeling PY - 2024 AN - OPUS4-61691 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Riedel, Soraya T1 - Tracing toxic fumonisins - Fumonisin sensing by Aspergillus niger Fumonisin Amine Oxidase (AnFAO) and amperometric hydrogen peroxide detection N2 - Fumonisins are a class of toxic secondary metabolites produced by various Fusarium species. The two most important producers of fumonisins are F. verticillioides and F. proliferatum but also Aspergillus niger is known to produce fumonisins. Most frequently they occur on maize, but also other grains can be contaminated with this group of mycotoxins. Exposure to fumonisins by dietary intake can have serious health effects on farm animals such as equine leukoencephalomalacia and porcine pulmonary oedema and is associated with neural tube defects and esophageal cancer in humans. Thus, the European Commission sets legal limits for fumonisins in foodstuffs. The detection of fumonisins is frequently performed in laboratories by chromatographic methods, which are costly and require trained personnel. Simplifying the analysis is therefore a major goal using portable detection systems. Electrochemical enzymatic biosensors offer great promise to meet this demand. Here we report for the first time an enzymatic fumonisin sensing approach with amperometric detection. For this purpose, an Aspergillus niger fumonisin amine oxidase (AnFAO) catalyzing the oxidative deamination of fumonisins, producing hydrogen peroxide, was recombinantly produced in E. coli. For the first time, the specific enzyme activity of AnFAO was determined using a horseradish peroxidase-based fluorescence assay. It was found that the specific activity of AnFAO using 20 μM Fumonisin B1 as substrate is higher than for 20 μM Fumonisin B2 with 0.122 U mg-1 and 0.058 U mg-1, respectively. It was possible to show a dependence of enzyme activity with enzyme – and substrate-concentration. For fumonisin B1 detection, the enzyme was coupled covalently to magnetic particles and the enzymatically produced H2O2 was detected amperometrically in a flow injection system using Prussian blue carbon electrodes. The developed method allows to quantify fumonisin B1 concentrations down to 1.5 µM and demonstrates that the recombinantly produced AnFAO was able to deaminate different concentrations of fumonisin even in immobilized form. Thus, this enzyme is well suited to develop an enzyme based electrochemical biosensor for fumonisin contaminated food and feed. T2 - Posterschau Adlershofer Forschungsforum CY - Berlin, Germany DA - 11.11.2022 KW - Biosensor KW - Amperometry KW - Mycotoxins KW - Electrochemical sensor KW - Recombinant protein expression PY - 2022 N1 - Geburtsname von Riedel, Soraya: Höfs, S. - Birth name of Riedel, Soraya: Höfs, S. AN - OPUS4-56248 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Riedel, Soraya T1 - Application of 3,3’,5,5’-Tetramethylbenzidine (TMB) in Amperometric Immunoassays for Mycotoxin Detection N2 - Electrochemical methods make great promise to meet the demand for user-friendly on-site devices for monitoring important parameters. Food industry often runs own lab procedures, e.g., for mycotoxin analysis, but it is a major goal to simplify analysis, linking analytical methods with miniaturized technologies. Enzyme-linked immunosorbent assays, with photometric detection of the horseradish peroxidase (HRP) substrate 3,3’,5,5’-tetramethylbenzidine (TMB), form a good basis for sensitive detection. To provide a straight-forward approach for the miniaturization of the detection step, we have studied the pitfalls of the electrochemical TMB detection. By cyclic voltammetry, it could be shown that TMB electrochemistry is strongly dependent on the pH and the electrode material. It was found that screen-printed gold electrodes and a very low pH value (pH 1) are well-suited to perform the electrochemical detection of TMB, due to the reversible character of the redox reaction under these conditions. Under these conditions, a good signal stability over several measuring cycles is achieved, providing the basis for analyzing multiple samples. In contrast to this, for carbon screen-printed electrodes, it was found that the signal response has changed after the electrochemical reaction with TMB at pH 1. At moderately acidic conditions (pH 4), neither with carbon nor with gold electrodes a reproducible electrochemical detection of TMB could be achieved. Based on these findings, we created a smartphone-based, electrochemical, immunomagnetic assay for the detection of ochratoxin A (OTA) and ergometrine in food samples. A competitive assay is performed on magnetic beads using HRP and TMB/H2O2 to generate the signal. Enzymatically oxidized TMB is quantified after addition of H2SO4 by amperometry with screen-printed gold electrodes in a custom-made wall-jet flow cell. The results are in good correlation with the established photometric detection method, providing a solid basis for sensing of further analytes in HRP-based assays using the newly developed miniaturized smartphone-based, electrochemical, immunomagnetic assay. T2 - 73rd Annual Meeting of the ISE CY - Online meeting DA - 12.09.2022 KW - Cylic Voltammetry KW - Immunoassay KW - Mycotoxins KW - Amperometry KW - Electrochemistry PY - 2022 N1 - Geburtsname von Riedel, Soraya: Höfs, S. - Birth name of Riedel, Soraya: Höfs, S. AN - OPUS4-55793 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Riedel, Soraya T1 - How to trace a cereal killer? N2 - How to trace the "cereal" killer? Fungal secondary metabolites, aka mycotoxins, pose a severe health risk for humans and animals. Since their occurrence in food, such as cereal products, is inevitable, the detection is of considerable importance. I want to demonstrate how the trace analysis of mycotoxins in foodstuff can be performed outside the laboratory. For this purpose, sensitive, antibody-based methods are combined with electrochemical smartphone-based detection. T2 - Bionnale 2021 CY - Online meeting DA - 12.05.2021 KW - Mycotoxins KW - Electrochemistry KW - Ochratoxin A KW - Food safety PY - 2021 N1 - Geburtsname von Riedel, Soraya: Höfs, S. - Birth name of Riedel, Soraya: Höfs, S. AN - OPUS4-52658 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Hoffmann, A. T1 - Priority effects influence the production of mycotoxins of Fusarium and Alternaria N2 - Mycotoxigenic fungal pathogens Fusarium and Alternaria are a leading cause of loss in cereal production. On wheat-ears, they are confronted by bacterial antagonists such as pseudomonads. Studies on these groups’ interactions often neglect the infection process’s temporal aspects and the associated priority effects. In the present study, the focus was on how the first colonizer affects the subsequent ones. In a climate chamber experiment, wheat-ears were successively inoculated with two different strains (Alternaria tenuissima At625, Fusarium graminearum Fg23, or Pseudomonas simiae Ps9). Over three weeks, microbial abundances and mycotoxin concentrations were analyzed and visualized via Self Organizing Maps with Sammon Mapping (SOM-SM). All three strains revealed different characteristics and strategies to deal with co-inoculation: Fg23, as the first colonizer, suppressed the establishment of At625 and Ps9. Nevertheless, primary inoculation of At625 reduced all of the Fusarium toxins and stopped Ps9 from establishing. Ps9 showed priority effects in delaying and blocking the production of the fungal mycotoxins. The SOM-SM analysis visualized the competitive strengths: Fg23 ranked first, At625 second, Ps9 third. Our findings of species-specific priority effects in a natural environment and the role of the mycotoxins involved are relevant for developing biocontrol strategies. T2 - 42. Mycotoxin Workshop CY - Online meeting DA - 30.05.2021 KW - Microbe interactions KW - Antagonists KW - Mycotoxins PY - 2021 AN - OPUS4-52815 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -