TY - JOUR A1 - Jenning, M. A1 - Marklein, B. A1 - Ytterberg, J. A1 - Zubarev, R. A. A1 - Joshua, V. A1 - van Schaardenburg, D. A1 - van de Stadt, L. A1 - Catrina, A. I. A1 - Nonhoff, U. A1 - Häupl, T. A1 - Konthur, Zoltán A1 - Burmester, G. R. A1 - Skriner, K. T1 - Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection—a missing link for ACPA production N2 - Objectives: Porphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA. Methods: Recombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated. Results: RACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57-71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018). Conclusions: RACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry. KW - Zitrullin KW - Rheumatoide Arthritis KW - Proteinmodifikation KW - Autoantikörper PY - 2020 DO - https://doi.org/10.1136/annrheumdis-2019-216919 VL - 79 IS - 9 SP - 1194 EP - 1202 PB - BMJ Publishing Group Ltd AN - OPUS4-51372 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Marklein, B. A1 - Jenning, M. A1 - Konthur, Zoltán A1 - Häupl, T. A1 - Welzel, F. A1 - Nonhoff, U. A1 - Krobitsch, S. A1 - Mulder, D. M. A1 - Koenders, M. I. A1 - Joshua, V. A1 - Cope, A. P. A1 - Shlomchik, M. J. A1 - Anders, H.-J. A1 - Burmester, G. R. A1 - Hensvold, A. A1 - Catrina, A. I. A1 - Rönnelid, J. A1 - Steiner, G. A1 - Skriner, K. T1 - The citrullinated/native index of autoantibodies against hnRNP-DL predicts an individual "window of treatment success" in RA patients N2 - Background: There is a need for biomarker to identify patients “at risk” for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers. Methods: Using protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid Differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry. KW - Citrullinated KW - Rheumatoid arthritis KW - Protein-modification KW - Autoantibodies KW - Antigens PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-533682 DO - https://doi.org/10.1186/s13075-021-02603-x SN - 1478-6362 VL - 23 SP - 1 EP - 16 PB - BioMed Central CY - London AN - OPUS4-53368 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Marklein, B. A1 - Jenning, M. A1 - Konthur, Zoltán A1 - Häupl, T. A1 - Welzel, F. A1 - Nonhoff, U. A1 - Krobitsch, S. A1 - Mulder, D. M. A1 - Koenders, M. I. A1 - Joshua, V. A1 - Cope, A. P. A1 - Shlomchik, M. J. A1 - Anders, H.‑J. A1 - Burmester, G. R. A1 - Hensvold, A. A1 - Catrina, A. I. A1 - Rönnelid, J. A1 - Steiner, G. A1 - Skriner, K. T1 - Correction to: The citrullinated/native index of autoantibodies against hnRNP-DL predicts an individual "window of treatment success" in RA patients N2 - This is a corrigendum to the original article 'The citrullinated/native index of autoantibodies against hnRNP-DL predicts an individual "window of treatment success" in RA patients' that was published in the journal "Arthritis research & therapy", vol. 23 (2021), article no. 239. PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-542698 DO - https://doi.org/10.1186/s13075-021-02639-z SN - 1478-6362 VL - 23 SP - 1 EP - 2 PB - BioMed Central CY - London AN - OPUS4-54269 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Konthur, Zoltán T1 - Utilizing Aspergillus niger Fumonisin Amine Oxidase for the Electrochemical Detection of Fumonisin 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 and also within humans. Thus, the European Commission sets legal limits for fumonisins in feed and 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. 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 - Affinity 2023 - the 25th meeting of the International Society for Molecular Recognition CY - Lisbon, Portugal DA - 05.06.2023 KW - Mycotoxin KW - Amperometry KW - Biosensor KW - Food analysis PY - 2023 N1 - Geburtsname von Riedel, Soraya: Höfs, S. - Birth name of Riedel, Soraya: Höfs, S. AN - OPUS4-57717 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Konthur, Zoltán T1 - Deutschlandweite Bestimmung anthropogener Marker im Abwasser N2 - Die von der Europäischen Kommission im Jahr 2021 empfohlene, systematische Abwasserüberwachung mittels PCR-Techniken wird als ein wirksames Früh-warnsystem für künftige Pandemien durch Viren und andere Krankheitserreger betrachtet. Ein generelles Problem bei jeder quantitativen Messung von Parametern im Abwasser ist die starke Schwankung des Abwasservolumens und seiner Zusammensetzung im Tages- und Jahreszeitenverlauf. Um die Konzentrationsbestimmungen von Krankheitserregern im Abwasser in Korrelation mit menschlichen Einträgen setzen zu können, könnten sog. "anthropogene Marker" eine herausragende Rolle spielen, deren Eintrag ins Abwasser menschlichen Ursprungs sind. Im Projekt werden mittels ELISA die Konzentrationen ausgewählter anthropogener Marker in deutschlandweit gesammelten Abwasserproben erhoben und analysiert. T2 - Spurenstoffe und Krankheitserreger im Wasserkreislauf CY - Frankfurt/Main, Germany DA - 27.03.2023 KW - Carbamazepin KW - Koffein KW - Diclofenac KW - Isolithocholsäure PY - 2023 AN - OPUS4-57718 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CHAP A1 - Ch’ng, A. C. W. A1 - Konthur, Zoltán A1 - Lim, T. S. T1 - Magnetic Nanoparticle-Based Semi-automated Panning for High-Throughput Antibody Selection N2 - Bio-panning is a common process involved in recombinant antibody selection against defined targets. The biopanning process aims to isolate specific antibodies against an antigen via affinity selection from a phage display library. In general, antigens are immobilized on solid surfaces such as polystyrene plastic, magnetic beads, and nitrocellulose. For high-throughput selection, semi-automated panning selection allows simul-taneous panning against multiple target antigens adapting automated particle processing systems such as the KingFisher Flex. The system setup allows for minimal human intervention for pre-and post-panning steps such as antigen immobilization, phage rescue, and amplification. In addition, the platform is also adaptable to perform polyclonal and monoclonal ELISA for the evaluation process. This chapter will detail the protocols involved from the selection stage until the monoclonal ELISA evaluation with important notes attached at the end of this chapter for optimization and troubleshooting purposes. KW - Phage display KW - Monoclonal antibody KW - Antibody library PY - 2023 DO - https://doi.org/10.1007/978-1-0716-3381-6_15 VL - 2702 SP - 291 EP - 313 PB - Springer US CY - New York, NY AN - OPUS4-58359 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Konthur, Zoltán T1 - Tracking Human-Derived Contaminants in Wastewater: ELISA-Based Detection of Anthropogenic Markers in Samples from German Treatment Plants N2 - Wastewater-based epidemiology (WBE) plays a key role in pandemic management as an early warning system and a tool for guiding interventions. In Germany, combined sewer systems are prevalent, leading to fluctuations in wastewater volume and composition due to rainwater inflow, especially through street drains. This variability complicates the quantitative measurement of pathogens and pollutants in wastewater. While human excretion provides constant inputs, these parameters are often non-specific, lost, or degraded during transport. In contrast, pharmaceuticals excreted by humans, such as carbamazepine and diclofenac, as well as consumed caffeine, are fairly stable and serve as "anthropogenic markers." The simultaneous detection of these markers aids in normalizing WBE studies. Antibody-based methods, particularly Enzyme-Linked Immunosorbent Assays (ELISA), offer a cost-effective alternative to complex chromatographic techniques for monitoring anthropogenic markers. They demonstrate superior normalization quality compared to the frequently used quantitative PCR-based marker Pepper mild mottle virus (PMMoV). T2 - ANAKON 2025 CY - Leipzig, Germany DA - 10.03.2025 KW - Immunoassay KW - Spurenstoffe KW - Carbamazepin KW - anthropogener Marker KW - Koffein PY - 2025 AN - OPUS4-63494 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Konthur, Zoltán T1 - Enzyme-Based Electrochemical Detection of Fumonisins Applying Amperometric Sensing N2 - Fumonisins represent a class of toxic secondary metabolites produced primarily by Fusarium species, notably F. verticillioides and F. proliferatum, though Aspergillus niger can also produce these mycotoxins. They are most commonly found contaminating maize, but other grains are also at risk. Dietary exposure to fumonisins poses serious health risks to both humans and livestock, prompting the European Commission to set regulatory limits on their presence in food and animal feed. Traditional detection methods, such as chromatography, are often costly, are laboratory based and require specialized expertise. To address the need for simpler, more accessible detection methods, portable electrochemical biosensors present a promising solution. In this study, we introduce a novel enzymatic approach for fumonisin detection using amperometric sensing. Here, a recently discovered fumonisin amine oxidase (AnFAO) from Aspergillus niger, which catalyses the oxidative deamination of fumonisins and generates hydrogen peroxide, was produced recombinantly in Escherichia coli. In our hands, the enzyme exhibited higher specific activity toward Fumonisin B1 compared to Fumonisin B2 at the same substrate concentration (0.0037 U/mg and 0.0014 U/mg at 25 µM substrate, respectively). We demonstrated that enzyme activity correlates with both enzyme and substrate concentrations. To detect Fumonisin B1, the enzyme was covalently attached to magnetic particles, and hydrogen peroxide production was measured amperometrically in a flow injection system with Prussian blue carbon electrodes. This method enabled the quantification of Fumonisin B1 concentrations as low as 1.5 µM and proved that recombinant AnFAO effectively deaminated fumonisins even in its immobilized form. These results highlight AnFAO’s potential for developing an enzyme-based electrochemical biosensor to detect fumonisin contamination in food and feed products. T2 - ANAKON 2025 CY - Leipzig, Germany DA - 10.03.2025 KW - Immunoassay KW - Mycotoxin KW - Electrochemistry KW - Antibody KW - Biosensor PY - 2025 AN - OPUS4-63496 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schneider, Rudolf A1 - Konthur, Zoltán T1 - Bestimmung von anthropogenen Markerkonzentrationen im Abwasser mittels eines antikörperbasierten Verfahrens (ELISA) N2 - Im Rahmen des AMELAG Projektes wurden im Auftrag des Umweltbundesamtes (VH1804) unterschiedliche anthropogene Marker in Abwasserproben aus Brandenburg und Mecklenburg-Vorpommern gemessen. In dieser Präsentation wurden einige Ergebnisse vorgestellt. T2 - AMELAG Abschlussveranstaltung am Robort-Koch-Institut CY - Berlin, Germany DA - 13.12.2024 KW - Östron KW - Koffein KW - Carbamazepin KW - Clarithromycin PY - 2024 AN - OPUS4-62194 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Konthur, Zoltán T1 - Bundesweite Bestimmung anthropogener Marker im Rohabwasser mittels Immunoassays N2 - Die von der Europäischen Kommission im Jahr 2021 empfohlene, systematische Abwasserüberwachung mittels PCR-Techniken wird als ein wirksames Frühwarnsystem für künftige Pandemien durch Viren und andere Krankheitserreger betrachtet. Ein generelles Problem bei jeder quantitativen Messung von Parametern im Abwasser ist die starke Schwankung des Abwasservolumens und seiner Zusammensetzung im Tages- und Jahreszeitenverlauf. Um die Konzentrationsbestimmungen von Krankheitserregern im Abwasser in Korrelation mit menschlichen Einträgen setzen zu können, könnten sog. "anthropogene Marker" eine herausragende Rolle spielen, deren Eintrag ins Abwasser menschlichen Ursprungs sind. Im Projekt werden mittels ELISA die Konzentrationen ausgewählter anthropogener Marker in deutschlandweit gesammelten Abwasserproben erhoben und analysiert. Die Messergebnisse zeigen eindeutige Standortunterschiede auch in Abhängigkeit der Kläranlagengröße und vorhandener Indirekteinleiter. Die Untersuchungen zeigen, dass nicht alle anthropogene Marker an allen Standorten für eine Normalisierung von PCR-Daten für epidemiologische Untersuchungen verwendbar sind. T2 - Wasser 2024 (Jahrestagung der Wasserchemischen Gesellschaft der GDCh) CY - Limburg an der Lahn, Germany DA - 06.05.2024 KW - Diclofenac KW - Koffein KW - Carbamazepin KW - Isolithocholsäure KW - Immunoassay PY - 2024 AN - OPUS4-60991 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -