TY - JOUR A1 - Mi, W. A1 - Josephs, R. D. A1 - Melanson, J. E. A1 - Dai, X. A1 - Wang, Y. A1 - Zhai, R. A1 - Chu, Z. A1 - Fang, X. A1 - Thibeault, M.-P. A1 - Stocks, B. B. A1 - Meija, J. A1 - Bedu, M. A1 - Martos, G. A1 - Westwood, S. A1 - Wielgosz, R. I. A1 - Liu, Q. A1 - Teo, T. L. A1 - Liu, H. A1 - Tan, Y. J. A1 - Öztuğ, M. A1 - Saban, E. A1 - Kinumi, T. A1 - Saikusa, K. A1 - Schneider, Rudolf A1 - Weller, Michael G. A1 - Konthur, Zoltán A1 - Jaeger, Carsten A1 - Quaglia, M. A1 - Mussell, C. A1 - Drinkwater, G. A1 - Giangrande, C. A1 - Vaneeckhoutte, H. A1 - Boeuf, A. A1 - Delatour, V. A1 - Lee, J. E. A1 - O'Connor, G. A1 - Ohlendorf, R. A1 - Henrion, A. A1 - Beltrão, P. J. A1 - Naressi Scapin, S. M. A1 - Sade, Y. B. T1 - PAWG Pilot Study on Quantification of SARS-CoV-2 Monoclonal Antibody - Part 1 N2 - Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a pilot study, CCQM-P216, was coordinated by the Chinese National Institute of Metrology (NIM), National Research Council of Canada (NRC) and the Bureau International des Poids et Mesures (BIPM). Eleven Metrology Institutes or Designated Institutes and the BIPM participated in the first phase of the pilot study (Part 1). The purpose of this pilot study was to develop measurement capabilities for larger proteins using a recombinant humanized IgG monoclonal antibody against Spike glycoprotein of SARS-CoV-2 (Anti-S IgG mAb) in solution. The first phase of the study was designed to employ established methods that had been previously studies by the CCQM Protein Analysis Working Group, involving the digestion of protein down to the peptide or amino acid level. The global coronavirus pandemic has also led to increased focus on antibody quantitation methods. IgG are among the immunoglobulins produced by the immune system to provide protection against SARS-CoV-2. Anti-SARS-CoV-2 IgG can therefore be detected in samples from affected patients. Antibody tests can show whether a person has been exposed to the SARS-CoV-2, and whether or not they potentially show lasting immunity to the disease. With the constant spread of the virus and the high pressure of re-opening economies, antibody testing plays a critical role in the fight against COVID-19 by helping healthcare professionals to identify individuals who have developed an immune response, either via vaccination or exposure to the virus. Many countries have launched large-scale antibody testing for COVID-19. The development of measurement standards for the antibody detection of SARS-CoV-2 is critically important to deal with the challenges of the COVID-19 pandemic. In this study, the SARS-CoV-2 monoclonal antibody is being used as a model system to build capacity in methods that can be used in antibody quantification. Amino acid reference values with corresponding expanded uncertainty of 36.10 ± 1.55 mg/kg, 38.75 ± 1.45 mg/kg, 18.46 ± 0.78 mg/kg, 16.20 ± 0.67 mg/kg and 30.61 ± 1.30 mg/kg have been established for leucine, valine, phenylalanine, isoleucine and proline, respectively. Agreement between nearly all laboratories was achieved for the amino acid analysis within 2 to 2.5 %, with one participant achieving markedly higher results due to a technical issue found in their procedure; this result was thus excluded from the reference value calculations. The relatively good agreement within a laboratory between different amino acids was not dissimilar to previous results for peptides or small proteins, indicating that factors such as hydrolysis conditions and calibration procedures could be the largest sources of variability. Peptide reference values with corresponding expanded uncertainty of 4.99 ± 0.28 mg/kg and 6.83 ± 0.65 mg/kg have been established for ALPAPIEK and GPSVFPLAPSSK, respectively. Not surprisingly due to prior knowledge from previous studies on peptide quantitation, agreement between laboratories for the peptide-based analysis was slightly poorer at 3 to 5 %, with one laboratory's result excluded for the peptide GPSVFPLAPSSK. Again, this level of agreement was not significantly poorer than that achieved in previous studies with smaller or less complex proteins. To reach the main text of this paper, click on Final Report. KW - Antibody quantification KW - Amino acid analysis KW - Peptide analysis KW - Round robin test PY - 2021 DO - https://doi.org/10.1088/0026-1394/59/1a/08001 VL - 59 IS - 1A SP - 08001 AN - OPUS4-54972 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Tscheuschner, Georg A1 - Kaiser, Melanie N. A1 - Lisec, Jan A1 - Beslic, D. A1 - Muth, Thilo A1 - Krüger, M. A1 - Mages, H.-W. A1 - Dorner, B. G. A1 - Knospe, J. A1 - Schenk, J. A. A1 - Sellrie, F. A1 - Weller, Michael G. T1 - MALDI-TOF-MS-based identification of monoclonal murine anti-SARS-CoV-2 antibodies within one hour N2 - During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used thirty-five monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied onto the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 45 minutes and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0 (https://gets.shinyapps.io/ABID/). This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context. KW - SARS-CoV-2 antibody KW - Reproducibility crisis KW - Peptide mass fingerprinting KW - Monoclonal antibody KW - Traceability KW - Antibody identification KW - Identity KW - Antibody light chain KW - MALDI-TOF-MS KW - Trypsin KW - Acidic cleavage KW - Antibody subclass KW - Database KW - Peak overlap KW - ABID KW - Sulfuric acid KW - Online software KW - Sequencing KW - Peptide coverage PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-545182 DO - https://doi.org/10.20944/preprints202203.0229.v1 SN - 2310-287X SP - 1 EP - 24 PB - MDPI CY - Basel AN - OPUS4-54518 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Steglich, P. A1 - Rabus, D. G. A1 - Sada, C. A1 - Paul, Martin A1 - Weller, Michael G. A1 - Mai, C. A1 - Mai, A. T1 - Silicon Photonic Micro-Ring Resonators for Chemical and Biological Sensing: A Tutorial N2 - Silicon photonic micro-ring resonators (MRR) developed on the silicon-on-insulator (SOI) platform, owing to their high sensitivity and small footprint, show great potential for many chemical and biological sensing applications such as label-free detection in environmental monitoring, biomedical engineering, and food analysis. In this tutorial, we provide the theoretical background and give design guidelines for SOI-based MRR as well as examples of surface functionalization procedures for label-free detection of molecules. After introducing the advantages and perspectives of MRR, fundamentals of MRR are described in detail, followed by an introduction to the fabrication methods, which are based on a complementary metal-oxide semiconductor (CMOS) technology. Optimization of MRR for chemical and biological sensing is provided, with special emphasis on the optimization of waveguide geometry. At this point, the difference between chemical bulk sensing and label-free surface sensing is explained, and definitions like waveguide sensitivity, ring sensitivity, overall sensitivity as well as the limit of detection (LoD) of MRR are introduced. Further, we show and explain chemical bulk sensing of sodium chloride (NaCl) in water and provide a recipe for label-free surface sensing. KW - Lab on a chip KW - Biosensor KW - Cmos KW - Silanization KW - Surface derivatization KW - Evanescent wave PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-529360 DO - https://doi.org/10.36227/techrxiv.14909901.v1 SP - 1 EP - 18 PB - IEEE CY - Piscataway Township AN - OPUS4-52936 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nerger, Tino A1 - Neumann, Patrick P. A1 - Weller, Michael G. T1 - A New Approach: Passive Smart Dust for Detection of Hazardous Substances N2 - Remote sensing of hazardous substances is a key task that can be achieved with the help of remotely operated platforms equipped with specific sensors. A huge variety of methods and used vehicles have been developed for different purposes in recent years. The term smart dust refers to a science fiction novel and develop shortly after into a research proposal at UC Berkley funded by DARPA. Subsequently, the topic gained attraction but was overall considered as to complex for the technologies available at that time. In the launched passive smart dust project, we shift to a simple “chemical intelligent” passive sensor particle on the ground combined a read-out active sensor attached to an Unmanned Aerial Vehicle (UAV). The reactive particle surface can be preadjusted in the lab for exact desired properties regarding certain reactions to hazardous substances. Moreover, the aimed interaction with the active sensor can be modified. Planed applications allow for different materials e.g., for short time measurement, being ecologically degradable, or weather stable for long time monitoring. T2 - 38th Danubia-Adria Symposium on Advances in Experimental Mechanics CY - Poros, Greece DA - 20.09.2022 KW - Smart Dust KW - Drone KW - Remote Detection KW - Hazardous substances PY - 2022 SP - 1 EP - 2 CY - Athens, Greece AN - OPUS4-55925 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Döring, Sarah A1 - Weller, Michael G. A1 - Reinders, Yvonne A1 - Konthur, Zoltán A1 - Jaeger, Carsten T1 - Challenges and Insights in Absolute Quantification of Recombinant Therapeutic Antibodies by Mass Spectrometry: An Introductory Review N2 - This review describes mass spectrometry (MS)-based approaches for the absolute quantification of therapeutic monoclonal antibodies (mAbs), focusing on technical challenges in sample treatment and calibration. Therapeutic mAbs are crucial for treating cancer, inflammatory, infectious, and autoimmune diseases. We trace their development from hybridoma technology and the first murine mAbs in 1975 to today’s chimeric and fully human mAbs. With increasing commercial relevance, absolute quantification of mAbs, traceable to SI units, has attracted attention from science, industry, and national metrology institutes (NMIs). Quantification of proteotypic peptides after enzymatic digestion using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as the most viable strategy, though methods targeting intact mAbs are still being explored. We review peptide-based quantification, focusing on critical experimental steps like denaturation, reduction, alkylation, choice of digestion enzyme, and selection of signature peptides. Challenges in amino acid analysis (AAA) for quantifying pure mAbs and peptide calibrators, along with software tools for targeted MS data analysis, are also discussed. Short explanations within each chapter provide newcomers an overview of the field’s challenges. Finally, we discuss prospects and limitations of developing standardized protocols and certified reference materials (CRMs) and suggest future applications of newer technologies for the absolute quantification of therapeutic antibodies. KW - Antibody KW - Mass spectrometry KW - Absolute quantification KW - Metrology KW - Reference products PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-621841 DO - https://doi.org/10.20944/preprints202412.1081.v1 SP - 1 EP - 22 PB - MDPI AN - OPUS4-62184 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Döring, Sarah A1 - Wulfes, Birte S. A1 - Atanasova, Aleksandra A1 - Jaeger, Carsten A1 - Walzel, Leopold A1 - Tscheuschner, Georg A1 - Flemig, Sabine A1 - Gawlitza, Kornelia A1 - Feldmann, Ines A1 - Konthur, Zoltán A1 - Weller, Michael G. T1 - Corundum Particles as Trypsin Carrier for Efficient Protein Digestion N2 - Reusable enzyme carriers are valuable for proteomic workflows, yet many supports are expensive or lack robustness. This study describes the covalent immobilization of recombinant trypsin on micrometer-sized corundum particles and assesses their performance in protein digestion and antibody analysis. The corundum surface was cleaned with potassium hydroxide, silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde. Recombinant trypsin was then attached, and the resulting imines were reduced with sodium cyanoborohydride. Aromatic amino acid analysis (AAAA) estimated an enzyme loading of approximately 1 µg/mg. Non-specific adsorption of human plasma proteins was suppressed by blocking residual aldehydes with a Tris-glycine-lysine buffer. Compared with free trypsin, immobilization shifted the temperature optimum from 50 to 60 °C and greatly improved stability in 1 M guanidinium hydrochloride. Activity remained above 80 % across several reuse cycles, and storage at 4 °C preserved functionality for weeks. When applied to digesting the NISTmAb, immobilized trypsin provided peptide yields and sequence coverage comparable to soluble enzyme and outperformed it at elevated temperatures. MALDI-TOF MS analysis of Herceptin digests yielded fingerprint spectra that correctly identified the antibody and achieved >60 % sequence coverage. The combination of low cost, robustness and analytical performance makes corundum-immobilized trypsin an attractive option for research and routine proteomic workflows. KW - Aluminum oxide KW - Mass spectrometry KW - Enzyme immobilization KW - Antibodies KW - Protein quantification PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-647944 DO - https://doi.org/10.20944/preprints202510.2002.v1 SP - 1 EP - 22 PB - Preprints.org AN - OPUS4-64794 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Nerger, Tino A1 - Neumann, Patrick P. A1 - Weller, Michael G. T1 - Drone-Based Localization of Hazardous Chemicals by Passive Smart Dust N2 - The distribution of tiny sensors over a specific area was first proposed in the late 1990s as a concept known as Smart Dust. Several efforts focused primarily on computing and networking capabilities but quickly ran into problems related to power supply, cost, data transmission, and environmental pollution. To overcome these limitations, we propose using paper-based (confetti-like) chemosensors that exploit the inherent selectivity of chemical reagents, such as colorimetric indicators. In this work, cheap and biodegradable passive sensors made from cellulose could successfully indicate the presence of hazardous chemicals, e.g., strong acids, by a significant color change. A conventional color digital camera attached to a drone could easily detect this from a safe distance. The collected data was processed to define the hazardous area. Our work presents a combination of the smart dust concept, chemosensing, paper-based sensor technology, and low-cost drones for flexible, sensitive, economical, and rapid detection of hazardous chemicals in high-risk scenarios. N2 - Die Verteilung winziger Sensoren über ein bestimmtes Gebiet wurde erstmals Ende der 1990er Jahre als Konzept namens „Smart Dust“ vorgeschlagen. Mehrere Bemühungen konzentrierten sich hauptsächlich auf Rechen- und Netzwerkfähigkeiten, stießen jedoch schnell auf Probleme im Zusammenhang mit der Stromversorgung, den Kosten, der Datenübertragung und der Umweltverschmutzung. Um diese Einschränkungen zu überwinden, schlagen wir die Verwendung von papierbasierten (konfettiartigen) Chemosensoren vor, die die inhärente Selektivität chemischer Reagenzien, wie z. B. kolorimetrischer Indikatoren, nutzen. In dieser Arbeit konnten günstige und biologisch abbaubare passive Sensoren aus Zellulose erfolgreich das Vorhandensein gefährlicher Chemikalien, z.B. starker Säuren, durch eine deutliche Farbänderung anzeigen. Eine herkömmliche Farb-Digitalkamera, die an einer Drohne befestigt ist, konnte dies aus sicherer Entfernung leicht erkennen. Die gesammelten Daten wurden verarbeitet, um den Gefahrenbereich zu abzugrenzen. Unsere Arbeit stellt eine Kombination aus dem Smart-Dust-Konzept, Chemosensorik, papierbasierter Sensortechnologie und kostengünstigen Drohnen für eine flexible, empfindliche, wirtschaftliche und schnelle Erkennung gefährlicher Chemikalien in Hochrisikoszenarien dar. KW - Confetti KW - Remote sensing KW - Drones KW - UAV KW - Optical detection KW - Chemosensor KW - pH indicator KW - Paper-based sensors KW - Harmful chemicals KW - Chemical desaster PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-621284 DO - https://doi.org/10.20944/preprints202408.0030.v1 SP - 1 EP - 17 PB - MDPI CY - Basle, Switzerland AN - OPUS4-62128 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Tannenberg, Robert A1 - Tscheuschner, Georg A1 - Raab, Christopher A1 - Flemig, Sabine A1 - Döring, Sarah A1 - Ponader, Marco A1 - Thurmann, Melinda A1 - Paul, Martin A1 - Weller, Michael G. T1 - Simplified Sample Preparation and Lateral Flow Immunoassay for the Detection of Plant Viruses N2 - Lateral flow immunoassays (LFA) are widely used for decentralized testing, but their application for in-field plant virus diagnostics is often limited by tedious sample preparation. Here, we present a simplified dipstick LFA for the detection and monitoring of cowpea chlorotic mottle virus (CCMV) as a model plant pathogen. The assay employes a monoclonal mouse antibody for capture and a polyclonal rabbit antibody conjugated to 80-nm gold nanoparticles for detection. Conventional sample and conjugate pads are omitted, allowing the test strips to be dipped directly into wells containing plant extract and antibody-gold conjugate. In addition, no plastic casing was necessary, which significantly reduces waste. It was shown that CCMV concentrations as low as 4 μg/L or 400 pg per sample could be reliably detected in 15 minutes. Specificity tests confirmed that other plant viruses, cowpea mosaic virus (CPMV) and tobacco mosaic virus (TMV), did not produce false positive results. Furthermore, we describe a field-compatible sampling procedure using a manual punch and a disposable syringe. This step combines sample grinding, extraction, and conjugate reconstitution within the syringe frit, enabling the analysis of punched leaf discs without laboratory equipment. When applied to CCMV-infected cowpea plants, the assay revealed systemic infection before visual symptoms became apparent. This work demonstrates that simplified LFAs combined with innovative sampling techniques can provide sensitive, specific, and rapid diagnostics for crop monitoring and support early intervention strategies in agriculture. N2 - Lateral Flow Immunoassays (LFA) werden häufig für dezentrale Tests verwendet, aber ihre Anwendung für die Diagnose von Pflanzenviren im Feld wird oft durch die mühsame Probenvorbereitung eingeschränkt. Hier stellen wir einen vereinfachten Dipstick-LFA zum Nachweis und zur Überwachung des Cowpea Chlorotic Mottle Virus (CCMV) als Modellpflanzenpathogen vor. Der Assay verwendet einen monoklonalen Maus-Antikörper zum Einfangen des Virus und einen polyklonalen Kaninchen-Antikörper, der an 80-nm-Goldnanopartikel konjugiert ist, zum Nachweis. Herkömmliche Proben- und Konjugatpads entfallen, sodass die Teststreifen direkt in Vertiefungen getaucht werden können, die Pflanzenextrakt und Antikörper-Gold-Konjugat enthalten. Darüber hinaus war keine Kunststoffhülle erforderlich, was den Abfall erheblich reduziert. Es zeigte sich, dass CCMV-Konzentrationen von nur 4 μg/L oder 400 pg pro Probe innerhalb von 15 Minuten zuverlässig nachgewiesen werden konnten. Spezifitätstests bestätigten, dass andere Pflanzenviren, das Cowpea Mosaic Virus (CPMV) und das Tobacco Mosaic Virus (TMV), keine falsch positiven Ergebnisse lieferten. Darüber hinaus beschreiben wir ein feldtaugliches Probenahmeverfahren unter Verwendung eines manuellen Stanzers und einer Einwegspritze. Dieser Schritt kombiniert das Zerkleinern der Probe, die Extraktion und die Rekonstitution des Konjugats innerhalb der Spritzenfritte und ermöglicht so die Analyse von ausgestanzten Blattscheiben ohne Laborausrüstung. Bei der Anwendung auf CCMV-infizierte Augenbohnenpflanzen zeigte der Test eine systemische Infektion, bevor visuelle Symptome sichtbar wurden. Diese Arbeit zeigt, dass vereinfachte LFAs in Kombination mit innovativen Probenahmetechniken eine sensitive, spezifische und schnelle Diagnostik für die Überwachung von Nutzpflanzen ermöglichen und frühzeitige Interventionsstrategien in der Landwirtschaft unterstützen können. KW - Vigna unguiculata KW - Cowpea KW - Sample preparation KW - Plant pathogen KW - Mobile detection KW - Crop monitoring KW - Plastic waste KW - Lateral flow immunoassay KW - Plant virus KW - Sample pad KW - Conjugate pad PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-651731 DO - https://doi.org/10.20944/preprints202512.1492.v1 SP - 1 EP - 14 PB - MDPI CY - Basle, Switzerland AN - OPUS4-65173 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Bosc-Bierne, Gaby A1 - Weller, Michael G. T1 - Investigation of Impurities in Peptide Pools N2 - Peptide pools are important research tools in different biomedical fields. They consist of a complex mixture of defined peptides, which places high demands on the production and quality control of these products. Previously it was shown that the combination of UHPLC with high-resolution mass-spectrometry (HRMS) is a fast and powerful method to confirm the relative concentration and the structural identity of all peptides expected to be in the pool. In this work, the additional information contained in the UV chromatograms and mass spectra is used to search for impurities due to synthesis by-products, degradation during storage and transportation and to identify possible analytical artifacts. It was shown that most impurities are only present in trace amounts and can be considered uncritical for most applications. The most frequent and perhaps unexpected impurities were homo- and heterodimers caused by the free cysteines contained in these peptide pools. Furthermore, pyroglutamate and aspartimide formation, deamidation, methionine oxidation, and amino acid deletions could be found. This list is not intended to be comprehensive, but rather a brief guide to quickly identify impurities and, in the long term, to suggest possible changes in the composition of the peptide pools to avoid such impurities by design or by special precautions. N2 - Peptidpools sind wichtige Forschungswerkzeuge in verschiedenen biomedizinischen Bereichen. Sie bestehen aus einer komplexen Mischung definierter Peptide, was hohe Anforderungen an die Produktion und Qualitätskontrolle dieser Produkte stellt. Zuvor wurde gezeigt, dass die Kombination von UHPLC mit hochauflösender Massenspektrometrie (HRMS) eine schnelle und leistungsstarke Methode ist, um die relative Konzentration und die strukturelle Identität aller Peptide zu bestätigen, die im Pool erwartet werden. In dieser Arbeit werden die zusätzlichen Informationen in den UV-Chromatogrammen und Massenspektren genutzt, um nach Verunreinigungen durch Synthesenebenprodukte, Abbau während der Lagerung und des Transports zu suchen und mögliche analytische Artefakte zu identifizieren. Es wurde gezeigt, dass die meisten Verunreinigungen nur in Spuren vorhanden sind und für die meisten Anwendungen als unkritisch angesehen werden können. Die häufigsten und vielleicht unerwartetsten Verunreinigungen waren Homo- und Heterodimere, die durch die in diesen Peptidpools enthaltenen freien Cysteine verursacht wurden. Außerdem konnten Pyroglutamat- und Aspartimidbildung, Deamidierung, Methioninoxidation und Aminosäurendeletion festgestellt werden. Diese Liste erhebt keinen Anspruch auf Vollständigkeit, sondern soll vielmehr eine kurze Anleitung zur schnellen Identifizierung von Verunreinigungen und langfristig zur Anregung möglicher Änderungen in der Zusammensetzung der Peptidpools sein, um solche Verunreinigungen durch Design oder besondere Vorsichtsmaßnahmen zu vermeiden. KW - Peptides KW - Mixtures KW - Degradation KW - Rearrangement KW - Cystein-Dimers KW - Immunology KW - MHC PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-621270 DO - https://doi.org/10.20944/preprints202412.0832.v1 SP - 1 EP - 16 PB - MDPI CY - Basle, Switzerland AN - OPUS4-62127 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nerger, Tino A1 - Neumann, Patrick P. A1 - Weller, Michael G. T1 - Passive Smart Dust For Detection Of Hazardous Substances N2 - High demand for remote sensing of hazardous substances. Possible solution: Use of distributed, low cost, and environmentally safe particles as passive sensors that can be read out remotely Chemical intelligence on the particle surface can be easily modified Particles enable optically quantifiable response and inference of target substances (also no maintenance or power supply required T2 - 38th Danubia-Adria Symposium on Advances in Experimental Mechanics CY - Poros, Greece DA - 20.09.2022 KW - Smart Dust KW - Drone KW - Remote Detection KW - Colorchanging Particles PY - 2022 AN - OPUS4-56025 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Tchipilov, Teodor A1 - Raysyan, Anna A1 - Weller, Michael G. T1 - Methods for the quantification of particle-bound protein – Application to reagents for lateral-flow immunoassays (LFIA) N2 - Protein immobilization for the functionalization of particles is used in various applications, including biosensors, lateral-flow immunoassays (LFIA), bead-based assays, and others. Common methods for the quantification of bound protein are measuring protein in the supernatant before and after coating and calculating the difference. This popular approach has the potential for a significant overestimation of the amount of immobilized protein since layers not directly bound to the surface (soft protein corona) are usually lost during washing and handling. Only the layer directly bound to the surface (hard corona) can be used in subsequent assays. A simplified amino acid analysis method based on acidic hydrolysis and RP-HPLC-FLD of tyrosine and phenylalanine (aromatic amino acid analysis, AAAA) is proposed to directly quantify protein bound to the surface of gold nano- and latex microparticles. The results are compared with indirect methods such as colorimetric protein assays, such as Bradford, bicinchoninic acid (BCA), as well as AAAA of the supernatant. For both particle types, these indirect quantification techniques show a protein overestimation of up to 1700% compared to the direct AAAA measurements. In addition, protein coating on latex particles was performed both passively through adsorption and covalently through EDC/sulfo-NHS chemistry. Our results showed no difference between the immobilization methodologies. This finding suggests that usual protein determination methods are no unambiguous proof of a covalent conjugation on particles or beads. KW - Soft protein corona KW - Hard protein corona KW - Gold particles KW - Nanoparticles KW - Mikroparticles KW - Antibody KW - Bioconjugation KW - Protein quantification KW - Supernatant KW - Sodium chloride method KW - Covalent conjugation KW - Latex particles KW - Lateral flow immunoassays PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-545365 DO - https://doi.org/10.20944/preprints202203.0332.v1 SP - 1 EP - 8 PB - MDPI CY - Basel AN - OPUS4-54536 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schroeder, Barbara A1 - Le Xuan, Hoa A1 - Völzke, Jule L. A1 - Weller, Michael G. T1 - Preactivation Crosslinking - An Efficient Method for the Oriented Immobilization of Antibodies N2 - Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications. KW - Antibody coating KW - Proximity-enhanced reaction KW - Immunoglobulins KW - IgG KW - Protein A KW - Protein G KW - Immunoprecipitation KW - Immunocapture KW - Regeneration KW - Biosensor KW - Immunosensor KW - Affinity chromatography KW - Immunoaffinity extraction KW - Oriented immobilization KW - Immunoassay KW - Bioconjugation PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-479786 DO - https://doi.org/10.3390/mps2020035 SN - 2409-9279 VL - 2 IS - 2 SP - 1 EP - 14 PB - MDPI CY - Basel, Switzerland AN - OPUS4-47978 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Schroeder, Barbara A1 - Le Xuan, Hoa A1 - Völzke, Jule L. A1 - Weller, Michael G. T1 - Preactivation crosslinking – An efficient method for the oriented immobilization of antibodies N2 - Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications. KW - Antibody coating KW - Proximity-enhanced reaction KW - Immunoglobulins KW - IgG KW - Protein G KW - Protein A KW - Immunoprecipitation KW - Immunocapture KW - Stabilization KW - Biosensor KW - Biochip KW - Microarray KW - ELISA KW - Immunoassay KW - Immunosensor KW - Crosslinker KW - Nanoparticles KW - Click chemistry KW - Herceptin KW - Trastuzumab PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-478797 DO - https://doi.org/10.20944/preprints201904.0205.v1 SN - 2310-287X SP - 1 EP - 17 PB - MDPI CY - Basel AN - OPUS4-47879 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Tscheuschner, Georg A1 - Schwaar, Timm A1 - Weller, Michael G. T1 - Fast Confirmation of Antibody Identity by MALDI-TOF-MS Fingerprints N2 - Thousands of antibodies for diagnostic and other analytical purposes are on the market. However, it is often difficult to identify duplicates, reagent changes, and to assign the correct original publications to an antibody. This slows down scientific progress and might even be a cause of irreproducible research and a waste of resources. Recently, activities were started to suggest the sole use of recombinant antibodies in combination with the open communication of their sequence. In this case, such uncertainties should be eliminated. Unfortunately, this approach seems to be rather a long-term vision since the development and manufacturing of recombinant antibodies remain quite expensive in the foreseeable future. Also, nearly all commercial antibody suppliers may be reluctant to publish the sequence of their antibodies, since they fear counterfeiting. De-novo sequencing of antibodies is also not feasible today for a reagent user without access to the hybridoma clone. Nevertheless, it seems to be crucial for any scientist to have the opportunity to identify an antibody undoubtedly to guarantee the traceability of any research activity using antibodies from a third party as a tool. For this purpose, we developed a method for the identification of antibodies based on a MALDI-TOF-MS fingerprint. To circumvent lengthy denaturation, reduction, alkylation, and enzymatic digestion steps, the fragmentation was performed with a simple formic acid hydrolysis step. Eighty-nine unknown monoclonal antibodies were used for this study to examine the feasibility of this approach. Although the molecular assignment of peaks was rarely possible, antibodies could be easily recognized in a blinded test, simply from their mass-spectral fingerprint. A general protocol is given, which could be used without any optimization to generate fingerprints for a database. We want to propose that in most scientific projects relying critically on antibody reagents, such a fingerprint should be established to prove and document the identity of the used antibodies and to assign a specific reagent to a datasheet of a commercial supplier, a public database record or an antibody ID. KW - Reproducibility KW - Quality Control KW - Traceability KW - Diagnostics KW - ELISA KW - Immunoassay PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-506590 DO - https://doi.org/10.20944/preprints202002.0207.v1 SN - 2310-287X SP - 1 EP - 17 PB - MDPI CY - Basel AN - OPUS4-50659 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Ponader, Marco A1 - Weller, Michael G. T1 - Affinitätschromatografie - Neues Trägermaterial aus Borosilikatglas N2 - Therapeutische Antikörper sind innerhalb weniger Jahre zur wichtigsten pharmazeutischen Produktklasse aufgestiegen. Für 2023 werden weltweite Umsätze von über 200 Milliarden USD erwartet. Auch diagnostische Antikörper sind mittlerweile unverzichtbare Produkte, auf deren Basis zahllose Immunoassays und andere Schnelltests entwickelt wurden. Neben der bereits sehr aufwendigen Herstellung von Antikörpern ist deren Aufreinigung aus komplexen Zellkulturmedien oder Blutseren und -plasmen zu einem Engpass in der Produktion und Nutzung dieser komplexen Proteine geworden. Schnelle und einfache Reinigungsmethoden für Antikörper sind daher sehr gefragt. KW - Affinitätschromatographie KW - Affinitätsextraktion KW - Antikörper KW - Glasmonolith KW - Additive Fertigung KW - IgG KW - Immunglobulin KW - HPLC KW - FPLC KW - biokompatibel KW - Borosilikatglas KW - gesintert KW - Titan-Halterung KW - Druckstabilität PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-477490 UR - https://www.chemiextra.com/ N1 - Geburtsname von Ponader, Marco: Wilke, M. - Birth name of Ponader, Marco: Wilke, M. IS - 4 SP - 16 EP - 17 PB - Sigwerb CY - Zug AN - OPUS4-47749 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nerger, Tino A1 - Neumann, Patrick P. A1 - Weller, Michael G. T1 - Drone-based Localization of Hazardous Chemicals by Passive Smart Dust N2 - We introduce a passive smart dust concept as a novel solution for environmental monitoring. Utilizing chemical reagents like colorimetric indicators and other chemosensors, these particles detect varying environmental conditions. We developed paper-based sensors that are both cost-effective and eco-friendly. In practical tests, these sensors, dispersed over a designated area, successfully identified hazardous substances by changing their color when exposed to acids or bases. This color change was remotely detectable using a drone-mounted color camera. The data thus obtained was processed through specialized software, accurately pinpointing areas of contamination. This method proves the efficacy and scalability of passive smart dust technology for real-time, environmentally sustainable remote sensing of hazardous materials T2 - 2024 IEEE International Symposium on Olfaction and Electronic Nose (ISOEN) CY - Grapevine, Texas, USA DA - 12.05.2024 KW - Indicator KW - Passive Smart Dust KW - Colorimetric Sensor KW - Drone KW - Cellulose KW - Environmental Monitoring KW - Confetti KW - Hazard Detection PY - 2024 SN - 979-8-3503-7053-9 VL - 1 SP - 1 EP - 3 PB - IEEE CY - Piscataway, NJ 08855-1331 USA AN - OPUS4-60176 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nerger, Tino A1 - Neumann, Patrick P. A1 - Weller, Michael G. T1 - Passive Smart Dust a versatile low-cost sensor platform N2 - Coated cellulose particles as colourimetric passive sensors that are detected by the optical camera system of a drone. In this way, hazardous substances, e.g. acids, can be detected from a safe distance in a cost-effective and environmentally friendly way. N2 - Beschichtete Zellulose Partikel als kolorimetrische passive Sensoren, die mittels optische Kamerasystem einer Drohne erfasst werden. Somit können Gefahrenstoffe, in ersten Versuchen zB Säuren aus sicherer Entfernung, kostengünstig und umweltverträglich detektiert werden. T2 - SMSI 2023 Sensor and Measurement Science International CY - Nuremberg, Germany DA - 08.05.2023 KW - Passive Smart Dust KW - Drone KW - Colorimetric sensor particles KW - Remote detection PY - 2023 DO - https://doi.org/10.5162/SMSI2023/C5.3 SP - 1 EP - 2 AN - OPUS4-57527 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Antibody quality - a never-ending story? N2 - Deficiencies in research antibody quality lead to economic losses up to several billions of Euros annually. Poorly documented antibody experiments lead to a flood of publications without any chance to be reproduced and without any scientific value.The dominance of antibody specifications in complex analytical procedures is often highly underestimated. Most journals and funding agencies lack the expertise to identify major flaws in antibody experiments and their documentation. Most companies sell insufficiently characterized and documented antibodies. Recombinant antibodies are not the solution for antibody validation. T2 - Seminar des Instituts für Analytische Chemie der Universität Wien CY - Wien, Austria DA - 17.11.2016 KW - Antibodies KW - Reproducibility KW - Crisis KW - Antibody ID PY - 2016 AN - OPUS4-38424 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Sauter, O. A1 - Neumann, Patrick P. A1 - Nerger, Tino A1 - Weller, Michael G. ED - Heß, M. T1 - Aus sicherer Entfernung BT - das Magazin für die Gefahrgut-Logistik N2 - Da chemische Gefahrstoffe im Boden eine ernsthafte Bedrohung für Leben und Gesundheit darstellen, das Grundwasser kontaminieren und langfristige Umweltschäden verursachen können, ist ihre frühzeitige Erkennung von großer Bedeutung. Um solche Gefahrstoffe aus sicherer Entfernung detektieren zu können, wurde bei der Bundesanstalt für Materialforschung und -prüfung (BAM) im Rahmen eines Forschungsvorhabens seit Anfang 2022 an einem neuen Ansatz zur Erkennung und Überwachung chemischer Gefahrstoffe gearbeitet. KW - Cellulose KW - Drohne KW - Kolorimetrische Sensoren KW - Passive Smart Dust PY - 2025 SN - 0016-5808 VL - 2025 IS - 8 SP - 26 EP - 28 PB - ecomed-Storck GmbH, Storck Verlag Hamburg CY - Hamburg AN - OPUS4-64109 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Tchipilov, Teodor A1 - Meyer, Klas A1 - Weller, Michael G. T1 - Quantitative 1H nuclear magnetic resonance (qNMR) of aromatic amino acids for protein quantification N2 - qNMR is a valuable technique for metrological studies due to the uniformity of its signal response for all chemical species of an isotope of interest, which enables compound-independent calibration. However, protein quantification remained challenging as large molecules produce wide, low-intensity signals that reduce the already low sensitivity. Combining qNMR with the hydrolysis of protein samples into amino acids circumvents many of these issues and facilitates the use of NMR spectroscopy for absolute protein and peptide quantification.In this work, different conditions have been tested for quantifying aromatic amino acids and proteins. First, we examined the pH-based signal shifts in the aromatic region. The preferable pH depends on the selection of the amino acids for quantification and which internal standard substance should be used to avoid peak overlap. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, have been applied as internal standards. The quantification of amino acids from an amino acid standard, as well as from a certified reference material (bovine serum albumin), was performed. Using the first two suggested internal standards, recovery was ~ 97 % for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98 ± 2 and 88 ± 4 %, respectively, at a protein concentration of 16 g/L or 250 µM. KW - Amino acid analysis KW - AAA KW - Protein hydrolysis KW - Metrology KW - Traceability KW - Reference materials KW - Internal standards KW - Calibration PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-564520 DO - https://doi.org/10.20944/preprints202211.0569.v1 SP - 1 EP - 13 PB - MDPI CY - Basel AN - OPUS4-56452 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Antibody-based SAW sensor for the detection of explosives N2 - A robust and sensitive method for the detection of the explosive trinitrotoluene (TNT) was developed. The detection limit was determined to be around 0.5 µg/L. The fast signal response of less than 1 minute shows that this approach is suitable for security and other time-critcal applications. In addition, the very low cross-reactivity highly reduces the number of false-positives in relation to competing techniques, including sniffer dogs. Due to the multianalyte ability of the SAW system, several explosives might be detected in parallel. T2 - BAM Meeting 2016 CY - Berlin, Germany DA - 17.02.2016 KW - TNT KW - Trinitrotoluene KW - Explosives KW - Airport security KW - High-speed biosensor KW - Selectivity KW - Sensitivity KW - Self-assembled monolayer KW - Gold surface KW - Immunosensor KW - Reversibility KW - Inhibition assay KW - Polyethylene glycol KW - PEG PY - 2016 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-366265 UR - http://f1000research.com/posters/5-1352 DO - https://doi.org/10.7490/f1000research.1112309.1 AN - OPUS4-36626 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Identification of toxicants in complex environmental samples using HPLC combined online with toxicity testing T2 - Anakon 2007 CY - Jena, Germany DA - 2007-03-27 PY - 2007 AN - OPUS4-14737 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Weller, Michael G. T1 - Das Phantom von Heilbronn - Eine Herausforderung für die Analytik? KW - Wattestäbchen KW - DNA KW - Spurensicherung KW - Qualitätskontrolle KW - Analytische Expertise PY - 2009 SN - 0939-0065 IS - 3 SP - 11 EP - 12 PB - Gesellschaft Deutscher Chemiker CY - Frankfurt, M. AN - OPUS4-19890 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Detection of explosives by bioanalytical techniques T2 - Potsdam Days on Bioanalysis 2011 CY - Potsdam, Germany DA - 2011-11-09 PY - 2011 AN - OPUS4-24952 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Baumgartner, S. A1 - Fürtler-Leitzenberger, I. A1 - Drs, E. A1 - Molinelli, A. A1 - Krska, R. A1 - Immer, U. A1 - Schmitt, K. A1 - Bremer, M. A1 - Haasnoot, W. A1 - Danks, C. A1 - Romkies, V. A1 - Reece, P. A1 - Wilson, P. A1 - Kiening, M. A1 - Weller, Michael G. A1 - Niessner, R. A1 - Corsini, E. A1 - Mendonca, S. ED - Darsa P. Siantar, ED - Mary W. Trucksess, ED - Peter M. Scott, ED - Eliot M. Herman, T1 - European survey for hidden allergens in food: A case study with peanut and hazelnut KW - Allergene KW - Erdnuß KW - Haselnuß KW - Allergentest KW - Lebensmittel KW - Kontamination PY - 2008 SN - 978-0-8412-6954-5 N1 - Serientitel: ACS Symposium Series – Series title: ACS Symposium Series SP - 370 EP - 381 AN - OPUS4-19052 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Weller, Michael G. T1 - Schnelle analytische Techniken für Screening und Monitoring KW - Immunsensoren KW - Immunoassays KW - Teststreifen KW - Probenahme PY - 2008 SN - 0939-0065 IS - 2 SP - 24 PB - Gesellschaft Deutscher Chemiker CY - Frankfurt, M. AN - OPUS4-17599 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Giesen, Charlotte A1 - Jakubowski, Norbert A1 - Panne, Ulrich A1 - Weller, Michael G. T1 - Ochratoxin-A-Bestimmung in Wein mittels eines ICP-MS-basierten Immunoassays PY - 2010 UR - http://www.analytik-news.de/Fachartikel/Volltext/BAM2.pdf SP - 1 EP - 6 PB - Dr. Beyer Internet-Beratung CY - Ober-Ramstadt AN - OPUS4-22702 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - One-bead-one-peptide libraries, their application, and other topics N2 - A novel method for screening one-bead-one-peptide libraries is presented and discussed. The approach comprises the following steps: 1. The peptide library is synthesized on peptide beads with a 4-(hydroxymethyl)benzoic acid (HMBA) linker. 2. The beads are placed on glass slides equipped with electrically conductive double-sided adhesive tape using a precision sieve. 3. The chip is incubated with fluorescence-labeled target molecules (e.g., antibodies or receptors) and scanned with a fluorescence scanner to identify the positive beads. 4. The peptides are cleaved from the polymer beads using ammonia gas. 5. The MALDI matrix is applied using a spray gun. 6. The positive beads are sequenced using MALDI-ToF-MS. T2 - Berlin-Delhi Network Symposium CY - Berlin, Germany DA - 31.10.2025 KW - Peptide KW - Aptamer KW - Bibliothek PY - 2025 AN - OPUS4-64562 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Steglich, P. A1 - Mai, C. A1 - Bondarenko, S. A1 - Paul, Martin A1 - Weller, Michael G. A1 - Schrader, S. A1 - Mai, A. T1 - BioPIC - Integration of Biosensors based on Photonic Integrated Circuits by Local-Backside Etching N2 - Silicon photonic sensors are promising candidates for lab-on-a-chip solutions with versatile applications and scalable production prospects using complementary metal-oxide semiconductor (CMOS) fabrication methods. However, the widespread use has been hindered because the sensing area adjoins optical and electrical components making packaging and sensor handling challenging. In this work, a local back-side release of the photonic sensor is employed, enabling a separation of the sensing area from the rest of the chip. This approach allows preserving the compatibility of photonic integrated circuits in the front-end of line and metal interconnects in the back-end of line. T2 - ATTRACT online Conference CY - Online meeting DA - 22.09.2020 KW - Silicon Photonics KW - Photonic Sensor KW - Photonic Integrated Circuits KW - Point-Of-Care-Diagnostics KW - CMOS KW - Microfluidics KW - Lab-on-a-chip KW - Ring resonator PY - 2020 UR - https://attract-eu.com/showroom/project/integration-of-biosensors-based-on-photonic-integrated-circuits-by-local-backside-etching-biopic/ SP - 1 EP - 5 AN - OPUS4-51735 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Methods for immunoaffinity extraction – Setup and characterization N2 - Affinity extraction is one of the most powerful separation methods. Regarded to be indispensable in some research fields, such as proteomics, the application in other fields is still scarce or even waning. Many researchers fear the perceived complexity, cost and unreliability of this approach. The presentation gives a broad overview of the most important methods and discusses some important points for their implementation. Having considered these aspects, it should be easier to establish an affinity separation approach to resolve challenging analytical questions. T2 - Seminar des Max-Planck-Instituts für Chemie (MPIC) CY - Mainz, Germany DA - 28.09.2016 KW - affinity chromatography KW - immunoaffinity KW - antibodies KW - sample preparation KW - bioanalysis PY - 2016 AN - OPUS4-37736 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Krop, U. A1 - Scherz, Franziska A1 - Weller, Michael G. A1 - Monks, K. T1 - Fast and robust purification of antibodies from human serum with a new monolithic protein A column N2 - The use of antibodies is becoming more and more important. Therewith the need for fast and robust methods for their purification and columns with a long lifetime is growing. In this application, we describe a fast and robust protein A affinity chromatography method with a new monolithic column. Antibodies were successfully purified in less than 1.5 minutes with a flowrate of 5 mL/min highlighting the advantages of this column and method. KW - Antibodies KW - Protein A KW - MNPQ KW - Technology transfer KW - Human serum KW - Purification KW - IgG KW - SDS PAGE KW - Regeneration PY - 2017 UR - http://www.knauer.net/en/application/fast_and_robust_purification_of_antibodies_from_human_serum_with_a_new_monolithic_protein_a_column.html SP - 1 EP - 2 PB - Knauer CY - Berlin AN - OPUS4-43163 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Bosc-Bierne, Gaby A1 - Ewald, Shireen A1 - Kreuzer, Oliver J. A1 - Weller, Michael G. T1 - Efficient Quality Control of Peptide Pools N2 - Peptide pools composed of short amino acid sequences have proven to be versatile tools in various research areas and clinical applications. They are powerful tools for epitope mapping, immunotherapy, and vaccine development. Their importance lies in their ability to map complex protein structures, enabling a comprehensive understanding of immune responses and facilitating the identification of potential therapeutic agents. The application of peptide pools also extends to the field of personalized medicine, offering tailored solutions for diseases such as cancer and infectious diseases. Peptide pools are complex mixtures of immunostimulatory antigens primarily intended for T-cell stimulation. They are commercially available in many different compositions and variants. However, unlike other reagents that consist of only one or a few compounds, peptide pools are highly complex products with limited stability. This makes their quality control a major challenge. Quantitative peptide analysis usually requires sophisticated methods, in most cases isotope-labeled standards and reference materials. None of these are routinely available for these products. Synthesis and purification of all labeled peptides might be required. Usually, this approach would be prohibitively laborious and expensive. Therefore, an approach is needed to provide a practical and feasible method for quality control of peptide pools. With insufficient quality control, the use of such products could lead to incorrect experimental results, which would worsen the well-known reproducibility crisis in the biomedical sciences. Here we propose the use of ultra-high performance liquid chromatography (UHPLC) with two detectors, a standard UV detector (at 214 nm) for quantitative analysis and a high-resolution mass spectrometer (HRMS) for identity confirmation. To be cost-efficient and fast, quantification and identification are performed in one chromatographic run. An optimized protocol is shown, and different peak integration methods are compared and discussed. This work was performed using a peptide pool known as CEF, which consists of 32 peptides derived from cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza virus, ranging from 8 to 12 amino acids in length. N2 - Peptidpools, die aus kurzen Aminosäuresequenzen bestehen, haben sich in verschiedenen Forschungsbereichen und klinischen Anwendungen als vielseitige Werkzeuge erwiesen. Sie sind leistungsstarke Werkzeuge für die Epitopkartierung, die Immuntherapie und die Impfstoffentwicklung. Ihre Bedeutung liegt in ihrer Fähigkeit, komplexe Proteinstrukturen zu kartieren, was ein umfassendes Verständnis der Immunantworten ermöglicht und die Identifizierung potenzieller Therapeutika erleichtert. Die Anwendung von Peptidpools erstreckt sich auch auf den Bereich der personalisierten Medizin und bietet maßgeschneiderte Lösungen für Krankheiten wie Krebs und Infektionskrankheiten. Peptidpools sind komplexe Mischungen immunstimulierender Antigene, die hauptsächlich zur T-Zell-Stimulation bestimmt sind. Sie sind in vielen verschiedenen Zusammensetzungen und Varianten im Handel erhältlich. Im Gegensatz zu anderen Reagenzien, die nur aus einer oder wenigen Verbindungen bestehen, sind Peptidpools jedoch hochkomplexe Produkte mit begrenzter Stabilität. Dies macht ihre Qualitätskontrolle zu einer großen Herausforderung. Die quantitative Peptidanalyse erfordert in der Regel ausgefeilte Methoden, in den meisten Fällen isotopenmarkierte Standards und Referenzmaterialien. Keines dieser Verfahren ist für diese Produkte routinemäßig verfügbar. Möglicherweise ist die Synthese und Reinigung aller markierten Peptide erforderlich. In der Regel wäre dieser Ansatz unerschwinglich aufwendig und teuer. Daher ist ein Ansatz erforderlich, der eine praktische und praktikable Methode für die Qualitätskontrolle von Peptidpools bietet. Bei unzureichender Qualitätskontrolle könnte die Verwendung solcher Produkte zu falschen Versuchsergebnissen führen, was die bekannte Reproduzierbarkeitskrise in den biomedizinischen Wissenschaften verschlimmern würde. Hier schlagen wir die Verwendung der Ultrahochleistungsflüssigkeitschromatographie (UHPLC) mit zwei Detektoren vor, einem Standard-UV-Detektor (bei 214 nm) für die quantitative Analyse und einem hochauflösenden Massenspektrometer (HRMS) für die Identitätsbestätigung. Um kosteneffizient und schnell zu sein, werden Quantifizierung und Identifizierung in einem chromatographischen Durchlauf durchgeführt. Es wird ein optimiertes Protokoll vorgestellt und verschiedene Peak-Integrationsmethoden werden verglichen und diskutiert. Diese Arbeit wurde mit einem Peptidpool namens CEF durchgeführt, der aus 32 Peptiden besteht, die vom Cytomegalovirus (CMV), Epstein-Barr-Virus (EBV) und Influenzavirus stammen und zwischen 8 und 12 Aminosäuren lang sind. KW - Quality control KW - UHPLC-UV-HRMS KW - Relative peptide quantification KW - Compound confirmation KW - Structure confirmation KW - Cost efficiency KW - Infectious diseases KW - Orbitrap PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-621307 DO - https://doi.org/10.20944/preprints202404.0992.v1 SN - 2310-287X SP - 1 EP - 18 PB - MDPI CY - Basel AN - OPUS4-62130 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Völzke, Jule L. A1 - Hodjat Shamami, Parya A1 - Gawlitza, Kornelia A1 - Feldmann, Ines A1 - Zimathies, Annett A1 - Weller, Michael G. T1 - High-purity corundum as support for affinity extractions from complex samples N2 - Nonporous corundum powder, known as an abrasive material in the industry, was functionalized covalently with protein binders to isolate and enrich specific proteins from complex matrices. The materials based on corundum were characterized by TEM, ESEM, BET, DLS, and zeta potential measurements. The strong Al-O-P bonds between the corundum surface and amino phosphonic acids are used to introduce functional groups for further conjugations. The common cross-linker glutaraldehyde was compared with a hyperbranched polyglycerol (PG) of around 10 kDa. The latter is oxidized with periodate to generate aldehyde groups that can covalently react with the amines of the surface and the amino groups from the protein via a reductive amination process. The amount of bound protein was quantified via aromatic amino acid analysis (AAAA). This work shows that oxidized polyglycerol can be used as an alternative to glutaraldehyde. With polyglycerol, more of the model protein bovine serum albumin (BSA) could be attached to the surface under the same conditions, and lower nonspecific binding (NSB) was observed. As a proof of concept, IgG was extracted with protein A from crude human plasma. The purity of the product was examined by SDS-PAGE. A binding capacity of 1.8 mg IgG per g of corundum powder was achieved. The advantages of corundum are the very low price, extremely high physical and chemical stability, pressure resistance, favorable binding kinetics, and flexible application. KW - Protein KW - Bioseparation KW - Purification KW - Immunoprecipitation KW - Affinity chromatography KW - Polyglycerol KW - Glutaraldehyde KW - Linker KW - Bioconjugation KW - Self-assembled monolayer (SAM) KW - Periodate oxidation KW - Reductive amination KW - Antibodies KW - Igg KW - Immunoglobulins KW - Carrier KW - Solid phase KW - Hyperbranched polymer KW - Aromatic amino acid analysis aaaa PY - 2022 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-555142 DO - https://doi.org/10.20944/preprints202208.0004.v1 SN - 2310-287X SP - 1 EP - 13 PB - MDPI CY - Basel AN - OPUS4-55514 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Paul, Martin A1 - Weller, Michael G. T1 - Antibody screening by microarray technology – Direct identification of selective high-affinity clones N2 - The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence a risky venture. We think that it is crucial to improve the screening process to eliminate most of the immanent deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and simultaneous performance of competition experiments. The latter can directly be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones and blank supernatant has been designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the IgG concentration, which is unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration is not possible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media had been used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system, we conclude that this approach should be preferable to most other protocols leading to many of false positives, causing expensive and lengthy confirmation steps to weed out the poor clones. KW - Hybridoma KW - Monoclonal Antibodies KW - Clones KW - Competitive Immunoassay KW - Hapten Immunoassay KW - False Positives PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-506621 DO - https://doi.org/10.20944/preprints201911.0023.v1 SN - 2310-287X SP - 1 EP - 17 PB - MDPI CY - Basel AN - OPUS4-50662 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Drone-Based Localization of Hazardous Chemicals by Passive Smart Dust N2 - Rapid detection and localization of hazardous chemicals is crucial for environmental monitoring, occupational safety, and emergency response. This study presents a novel approach that combines drone technology with passive smart dust – biodegradable, paper-based chemical sensors – to remotely identify and map hazardous substances. In contrast to conventional smart dust concepts based on electronic microsensors with significant limitations in power supply, cost and environmental impact, this method uses cellulose-based, confetti-like sensors impregnated with colorimetric or fluorescent indicators. These sensors change color in response to chemical exposure, enabling simple optical detection from a distance. A drone equipped with a conventional digital camera takes pictures of the deployed sensors, which are then analyzed by automated image processing. The system converts images from the red-green-blue color model (RGB) into hue-saturation-value space (HSV) and applies threshold filters and clustering algorithms to increase the accuracy of identifying hazardous zones. Field tests using thymol blue as a pH-sensitive indicator have successfully demonstrated the system's ability to detect and locate acid spills in an outdoor environment. The results confirmed that low-cost, off-the-shelf drones can effectively monitor affected areas by providing real-time data for responders while minimizing human exposure to hazardous chemicals. This approach offers a fast, scalable, cost-effective and environmentally friendly alternative to conventional hazard detection technologies. Other important advantages are the close proximity of the sensors to the contamination, the avoidance of downwash problems by the drone propellers and the generation of a dense mesh of data points. Since no complex electronic sensors are required and simple, biodegradable materials are used, the system is well suited for large-scale deployment in industrial accidents, environmental pollution and chemical disaster scenarios. Future research will investigate additional chemical indicators to expand detection capabilities, optimize sensor deployment, and refine data processing for greater accuracy. The passive smart dust system represents a new concept in remote chemical hazard monitoring and may have broad implications for accident response, environmental crime, facility security, and attacks with chemical weapons. N2 - Die schnelle Erkennung und Lokalisierung gefährlicher Chemikalien ist für die Umweltüberwachung, den Arbeitsschutz und den Katastrophenschutz von entscheidender Bedeutung. Diese Studie stellt einen neuartigen Ansatz vor, der Drohnen-Technologie mit Passivem Smart Dust – biologisch abbaubaren, papierbasierten chemischen Sensoren – kombiniert, um gefährliche Substanzen aus der Ferne zu identifizieren und zu kartieren. Im Gegensatz zu herkömmlichen Smart-Dust-Konzepten, die auf elektronischen Mikrosensoren mit erheblichen Einschränkungen bei der Stromversorgung, den Kosten und der Umweltbelastung basieren, verwendet diese Methode zellulosebasierte, konfetti-artige Sensoren, die mit kolorimetrischen oder fluoreszierenden Indikatoren imprägniert sind. Diese Sensoren ändern ihre Farbe bei Kontakt mit Chemikalien, sodass eine einfache optische Erkennung aus der Ferne möglich ist. Eine Drohne, die mit einer herkömmlichen Digitalkamera ausgestattet ist, nimmt Bilder der eingesetzten Sensoren auf, die dann durch automatisierte Bildverarbeitung analysiert werden. Das System konvertiert Bilder vom Rot-Grün-Blau-Farbmodell (RGB) in den Farbton-Sättigungs-Wert-Raum (HSV) und wendet Schwellenwertfilter und Clustering-Algorithmen an, um die Genauigkeit bei der Identifizierung gefährlicher Zonen zu erhöhen. Feldtests mit Thymolblau als pH-empfindlichem Indikator haben erfolgreich die Fähigkeit des Systems nachgewiesen, ausgelaufene Säure in Außenbereichen zu erkennen und zu lokalisieren. Die Ergebnisse bestätigen, dass kostengünstige, handelsübliche Drohnen betroffene Bereiche effektiv überwachen können, indem sie Einsatzkräften Echtzeitdaten liefern und gleichzeitig die Exposition von Menschen gegenüber gefährlichen Chemikalien minimieren. Dieser Ansatz bietet eine schnelle, skalierbare, kostengünstige und umweltfreundliche Alternative zu herkömmlichen Technologien zur Gefahrenerkennung. Weitere wichtige Vorteile sind die räumliche Nähe der Sensoren zur Kontamination, die Vermeidung von Abwindproblemen durch die Drohnenpropeller und die Erzeugung eines dichten Netzes von Datenpunkten. Da keine komplexen elektronischen Sensoren erforderlich sind und einfache, biologisch abbaubare Materialien verwendet werden, eignet sich das System gut für den großflächigen Einsatz bei Industrieunfällen, Umweltverschmutzung und chemischen Katastrophenszenarien. In zukünftigen Forschungsarbeiten sollen weitere chemische Indikatoren untersucht werden, um die Erkennungsmöglichkeiten zu erweitern, den Sensoreinsatz zu optimieren und die Datenverarbeitung für eine höhere Genauigkeit zu verfeinern. Das passive Smart-Dust-System stellt ein neues Konzept für die Fernüberwachung chemischer Gefahren dar und könnte weitreichende Auswirkungen auf die Unfallbekämpfung, Umweltkriminalität, Anlagensicherheit und Angriffe mit Chemiewaffen haben. T2 - 39th Session of the Scientific Advisory Board of the OPCW CY - The Hague, Netherlands DA - 01.04.2025 KW - Imaging KW - Remote Sensing KW - Chemosensors KW - Organization for the Prohibition of Chemical Weapons (OPCW) KW - CBRN Defence PY - 2025 AN - OPUS4-62897 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Challenges of protein analysis N2 - Selected Projects BAM Division 1.5 Test for sensitive SARS-CoV-2 detection Corundum as a novel affinity support Troponin biosensor based on chemiluminescence Plant virus purification (CCMV) Protein Quantification by 1H-NMR Ultra-miniaturized Biosensors N2 - Ausgewählte Projekte BAM-Abteilung 1.5 Test für empfindlichen SARS-CoV-2-Nachweis Korund als neuartige Affinitätscarriermaterialien Troponin-Biosensor auf Basis von Chemilumineszenz Reinigung von Pflanzenviren (CCMV) Proteinquantifizierung durch 1H-NMR Ultra-miniaturisierte Biosensoren T2 - Klausurtagung der MPI Abteilung „Multiphasenchemie“ CY - Mainz, Germany DA - 19.11.2024 KW - Corona test KW - Corundum KW - Affinity separation KW - Affinity chromatography KW - Troponin biosensor KW - Peptide aptamer KW - Cowpea chlorotic mottle virus KW - Nanoparticle KW - Nanocarrier KW - Nuclear magnetic resonance NMR KW - Protein quantification KW - HyPhox KW - Ring resonator PY - 2024 AN - OPUS4-62126 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Weller, Michael G. T1 - Referee Report 06 March 2017 N2 - This article is a valuable and welcome contribution to the ongoing discussion of antibody quality. The suggestion of a two-tier approach is helpful to distinguish between simple descriptive data and the validation of an antibody for a specified application. KW - Antibody KW - Validation PY - 2017 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-393333 UR - https://f1000research.com/articles/6-161/v1#referee-response-20349 DO - https://doi.org/10.5256/f1000research.11700.r20349 SN - 2046-1402 SP - 6 EP - 7 PB - F1000 Research Ltd. CY - London AN - OPUS4-39333 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Scherz, Franziska A1 - Krop, U. A1 - Monks, K. A1 - Weller, Michael G. T1 - Antikörperreinigung mit Glasmonolithen - Vereinfachung von Affinitätstrennungen mit HPLC-Systemen N2 - Druckstabile Glasmonolithen ermöglichen eine schnelle und unkomplizierte Reinigung von Antikörpern, z.B. aus Serum oder Zellkulturüberständen. Die sehr gute Regenerierbarkeit lässt eine lange Lebensdauer der Säulen erwarten, was die Kosten pro Probe niedrig hält. KW - Borosilicatglas KW - Immunglobuline KW - IgG KW - Serum KW - Plasma KW - Protein A KW - Protein G KW - Affinitätschromatographie KW - Agarose KW - Carrier-Material KW - Stationäre Phase KW - Immobilisierung KW - Highspeed KW - Trennung KW - Reinigung KW - Downstream Processing PY - 2018 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-445330 UR - http://www.git-labor.de/forschung/materialien/antikoerperreinigung-mit-glasmonolithen SN - 0016-3538 VL - 62 IS - 3 SP - 24 EP - 25 PB - Wiley-VCH Verlag GmbH & Co. KGaA CY - Weinheim AN - OPUS4-44533 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Weller, Michael G. T1 - Interview with Dr Michael G. Weller N2 - This author interview is by Dr Michael G. Weller, Head of Division 1.5 Protein Analysis at Federal Institute for Materials Research and Testing (BAM). BAM (www.bam.de/en) is a senior scientific and technical institute with responsibility to the German Federal Ministry for Economic Affairs and Energy. Dr Weller's review paper, Quality issues of research antibodies is available for download in Analytical Chemistry Insights. KW - Antibodies KW - Quality KW - Reproducibility PY - 2016 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-392326 UR - http://insights.sagepub.com/la_press_news.php?pa=view&news_item_id=4025 SP - 1 PB - SAGE CY - London AN - OPUS4-39232 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Weller, Michael G. T1 - The mystery of homochirality on earth N2 - Homochirality is an obvious feature of life on Earth. On the other hand, extraterrestrial samples contain largely racemic compounds. The same is true for any common organic synthesis. Therefore, it has been a perplexing puzzle for decades how these racemates could have formed enantiomerically enriched fractions as a basis for the origin of homochiral life forms. Numerous hypotheses have been put forward as to how preferentially homochiral molecules could have formed and accumulated on Earth. In this article, it is shown that homochirality of the abiotic organic pool at the time of formation of the first self-replicating molecules is not necessary and not even probable. It is proposed to abandon the notion of a molecular ensemble and to focus on the level of individual molecules. Although the formation of the first self-replicating, most likely homochiral molecule is a seemingly improbable event, on a closer look, it is almost inevitable that homochiral molecules have formed simply on a statistical basis. In this case, the non-selective leap to homochirality would be one of the first steps in chemical evolution directly out of a racemic “ocean”. Moreover, most studies focus on the chirality of the primordial monomers with respect to an asymmetric carbon atom. However, any polymer with a minimal size that allows folding to a secondary structure, would spontaneously lead to asymmetric higher structures (conformations). Most of the functions of these polymers would be influenced by this inherently asymmetric folding. To summarize, simple and universal mechanisms may have led to homochiral self-replicating systems in the context of chemical evolution. A homochiral monomer pool is deemed unnecessary and probably never existed on primordial Earth. N2 - Homochiralität ist ein offenkundiges Merkmal des Lebens auf der Erde. Andererseits enthalten extraterrestrische Proben überwiegend racemische Verbindungen. Das Gleiche gilt für jede gängige organische Synthese. Daher war es seit Jahrzehnten ein Rätsel, wie diese Racemate enantiomerenangereicherte Fraktionen als Grundlage für den Ursprung homochiraler Lebensformen bilden konnten. Es wurden zahlreiche Hypothesen darüber formuliert, wie sich bevorzugt homochirale Moleküle auf der Erde gebildet und angereichert haben könnten. In diesem Artikel wird gezeigt, dass die Homochiralität des abiotischen organischen Pools zum Zeitpunkt der Bildung der ersten selbstreplizierenden Moleküle nicht notwendig und nicht einmal wahrscheinlich ist. Es wird vorgeschlagen, die Vorstellung eines molekularen Ensembles aufzugeben und sich auf die Ebene der einzelnen Moleküle zu konzentrieren. Obwohl die Bildung des ersten selbstreplizierenden, vermutlich homochiralen Moleküls ein scheinbar unwahrscheinliches Ereignis ist, ist es bei näherer Betrachtung fast unvermeidlich, dass sich homochirale Moleküle einfach auf statistischer Basis gebildet haben. In diesem Fall wäre der nicht-selektive Sprung zur Homochiralität einer der ersten Schritte der chemischen Evolution direkt aus einem racemischen "Ozean" heraus. Außerdem konzentrieren sich die meisten Studien auf die Chiralität der ursprünglichen Monomere in Bezug auf ein asymmetrisches Kohlenstoffatom. Jedes Polymer mit einer Mindestgröße, die eine Faltung zu einer Sekundärstruktur ermöglicht, würde jedoch spontan zu asymmetrischen höheren Strukturen (Konformationen) führen. Die meisten Funktionen dieser Polymere würden durch diese inhärent asymmetrische Faltung beeinflusst. Zusammenfassend lässt sich sagen, dass einfache und universelle Mechanismen zu homochiralen selbstreplizierenden Systemen im Rahmen der chemischen Evolution geführt haben könnten. Ein homochiraler Monomerpool wird als unnötig erachtet und hat daher wahrscheinlich auf der Urerde auch nie existiert. KW - Chemical evolution KW - Abiogenesis KW - Chirality KW - Amino acids KW - Peptides KW - Folding chirality PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-598462 DO - https://doi.org/10.20944/preprints202401.1939.v1 SN - 2310-287X SP - 1 EP - 12 PB - MDPI CY - Basel AN - OPUS4-59846 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Weller, Michael G. T1 - Trennschärfe: Robustheit, Selektivität und Spezifität N2 - Zusammenfassung Selektivität 1. Widersprüchliche Definitionen in unterschiedlichen Bereichen 2. Analytische Definition (pragmatisch): Selektivität ist die Fähigkeit einer Methode zur Unterscheidung aller interessierender Stoffe in der Probe. 3. Tests: Blindwerte, Leerwerte, Blank Values; Peakform 4. Wichtige Einflussgrößen: Trennleistung, Auflösung, Peakkapazität, Trennkapazität 5. Verbesserung durch: Kopplungsverfahren, Änderung der chromatographischen „Selektivität“, Orthogonalität 6. Wichtig ist auch die Komplexität der Probe(n) 7. Selektivität in der Immunchemie/Biochemie wird oft als Kreuzreaktion gemessen. Zusammenfassung Robustheit 1. Sehr individuelle Kriterien – sinnvolle Auswahl notwendig! 2. Wichtige Testphasen: a)Änderung von Methodenparametern b)Stabilität über die Zeit c)Überführung in die Routine 3. Ringversuche bzw. Laborvergleichsmessungen 4. Wichtigstes Vergleichskriterium: Relative Verfahrensstandardabweichung 5. Extratipp: Maximale Diversität der Testproben sicherstellen (besonders bezüglich der Matrix) T2 - BAM Sommerschule 2016 "Qualitätssicherung in der analytischen Chemie" CY - Berlin, Germany DA - 04.07.2016 KW - Selektivität KW - Spezifität KW - Robustheit KW - Sommerschule KW - Blindwert KW - Peakkapazität KW - Kreuzreaktion PY - 2016 AN - OPUS4-37760 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Canning, S. A1 - Voskuil, J. A1 - Weller, Michael G. T1 - An author and reviewer in conversation – reproducibility and antibody validation N2 - A recent Opinion Article by Jan Voskuil (JV), Aeonian Biotech, discussed the challenges in validation of research antibodies. It was openly peer reviewed by Michael Weller (MW), Federal Institute for Materials Research and Testing (BAM), Germany. In this blog, both discuss these challenges, the reproducibility crisis and open data. DA - 19 May, 2017 KW - Antibodies KW - Monoclonal KW - Antibody ID KW - Quality control KW - Open data KW - Reproducibility KW - Peer review PY - 2017 UR - https://blog.f1000.com/2017/05/19/an-author-and-reviewer-in-conversation-reproducibility-and-antibody-validation SN - 2051-9796 SP - 1 EP - 5 PB - F1000 Research Ltd. CY - London AN - OPUS4-40310 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Weller, Michael G. A1 - Schneider, Rudolf A1 - Rurack, Knut T1 - Mit dem Testsystem zur Probe N2 - (Bio)chemische Sensoren ersetzen bei der Vor-Ort-Analytik die großen Messgeräte oder dienen dazu, Analyten im Verdachtsfall oder kontinuierlich zu überwachen. Das können pharmazeutische Stoffe im Abwasser sein, Aflatoxin in Milch oder Sprengstoff am Flughafen. Ein Überblick über Neuentwicklungen. KW - Biochemische Sensoren KW - Vor-Ort-Analytik KW - Schnelltests KW - Antikörper PY - 2021 DO - https://doi.org/10.1002/nadc.20214112170 VL - 69 IS - 10 SP - 71 EP - 74 PB - Wiley-VCH CY - Weinheim AN - OPUS4-54003 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Climent Terol, Estela A1 - Biyikal, Mustafa A1 - Gröninger, Delia A1 - Weller, Michael G. A1 - Martínez Mánez, R. A1 - Rurack, Knut T1 - Multiplex‐Nachweis von Analyten auf einem einzelnen Teststreifen mit Antikörper‐gesteuerten und Indikator freisetzenden mesoporösen Nanopartikeln N2 - Vor dem Hintergrund der einzigartigen Modularität, hohen Empfindlichkeit und Selektivität von Antikörper‐gesteuerten Indikatorfreisetzungssystemen (gAID‐Systemen) wurde hier ein Multiplex‐Assay für drei organische Explosivstoffmoleküle (TATP, TNT, PETN) entwickelt, der es erlaubt, die Analyten gleichzeitig in flüssiger Phase mit einem einzelnen Teststreifen und einem Fluoreszenzlesegerät bzw. Smartphone als Detektor in Konzentrationen bis in den unteren ppb‐Bereich in <5 min nachzuweisen. Alle drei Systeme, darunter die hier neu entwickelten Systeme für TNT und PETN, tolerieren zudem nicht nur gepufferte wässrige Modelllösungen, sondern auch komplexere Matrices. Neben einem konventionellen Teststreifen mit einem Kanal erlaubte uns die Anwendung von Wachsdrucktechnologie das Herstellen von mehrkanaligen Streifen mit vergleichbarer analytischer Leistungsfähigkeit, was das enorme Potenzial der modular aufgebauten, hybriden Biosensormaterialien im Hinblick auf eine für den Endanwender maßgeschneiderte Vor‐Ort‐Analytik unterstreicht. KW - Multiplex KW - Gesteuerten Nanopartikeln KW - Explosiven PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-518431 DO - https://doi.org/10.1002/ange.202009000 SN - 1521-3757 SN - 0044-8249 VL - 132 IS - 52 SP - 24071 EP - 24078 PB - Wiley-VCH CY - Weinheim AN - OPUS4-51843 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Fischer, Janina A1 - Kaufmann, Jan Ole A1 - Weller, Michael G. T1 - Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays N2 - The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this nformation, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols. N2 - Die Affinitätskonstante, auch bekannt als Gleichgewichtskonstante, Bindungskonstante, Gleichgewichtsassoziationskonstante oder der Kehrwert, die Gleichgewichtsdissoziationskonstante (Kd), kann als eine der wichtigsten Eigenschaften für jedes Antikörper-Antigen-Paar angesehen werden. Es wurden viele Methoden auf der Grundlage verschiedener Technologien vorgeschlagen und verwendet, um diesen Wert zu bestimmen. Da jedoch eine sehr große Anzahl von Veröffentlichungen und kommerziellen Datenblättern diese Informationen nicht enthalten, scheint es erhebliche Hindernisse bei der Durchführung solcher Messungen zu geben. In anderen Fällen, in denen solche Daten angegeben werden, haben sich die Ergebnisse oft als unzuverlässig erwiesen. Diese Situation könnte darauf hindeuten, dass die meisten der heute verfügbaren Technologien ein hohes Maß an Fachwissen erfordern, das in vielen Laboren nicht vorhanden zu sein scheint. In diesem Artikel stellen wir einen einfachen Ansatz vor, der auf der Standard-Immunoassay-Technologie basiert und einfach und schnell durchzuführen ist. Er beruht auf dem Effekt, dass sich der molare IC50-Wert dem Kd-Wert im Fall unendlich kleiner Konzentrationen der Reagenzienkonzentrationen nähert. Eine zweidimensionale Verdünnung der Reagenzien führt zu einer asymptotischen Konvergenz zu Kd. Der Ansatz weist eine gewisse Ähnlichkeit mit der bekannten Schachbrett-Titration auf, die zur Optimierung von Immunoassays verwendet wird. Ein bekannter Antikörper gegen das FLAG-Peptid, Klon M2, wurde als Modellsystem verwendet und die Ergebnisse wurden mit anderen Methoden verglichen. Dieser Ansatz kann in allen Fällen verwendet werden, in denen ein kompetitiver Test verfügbar ist oder entwickelt werden kann. Die Bestimmung einer Affinitätskonstante sollte zu den entscheidenden Parametern in jeder Qualitätskontrolle von Antikörper-bezogenen Produkten und Tests gehören und in Veröffentlichungen, die immunchemische Protokolle verwenden, obligatorisch sein. KW - ELISA KW - Microplate KW - Microtiter plate KW - IC50 KW - Test midpoint KW - Competitive immunoassay KW - Law of mass action KW - Thermodynamics KW - Equilibrium constant KW - SPR KW - Surface plasmon resonance KW - Clones KW - Interaction KW - kd value KW - ITC KW - MST PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-621298 DO - https://doi.org/10.20944/preprints202404.0996.v1 SN - 2310-287X SP - 1 EP - 11 PB - MDPI CY - Basel AN - OPUS4-62129 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Paul, Martin A1 - Tannenberg, Robert A1 - Tscheuschner, Georg A1 - Ponader, Marco A1 - Weller, Michael G. T1 - Cocaine detection by a laser-induced immunofluorometric biosensor N2 - The trafficking of illegal drugs by criminal networks at borders, harbors, or airports is an increasing issue in public health as these routes ensure the main supply of illegal drugs. The prevention of drug smuggling, including the installation of scanners and other analytical devices to detect ultra-small traces of drugs within a reasonable time frame, remains a challenge. The presented immunosensor is based on a monolithic affinity column with a large excess of immobilized hapten, which traps fluorescently labeled antibodies as long as the analyte cocaine is absent. In the presence of the drug, some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and microfluidic chip-based mixing devices and flow cells. The biosensor achieved limits of detection of 23 pM (7 ppt) of cocaine with a response time of 90 seconds and a total assay time below 3 minutes. With surface wipe sampling, the biosensor was able to detect 300 pg of cocaine. This immunosensor belongs to the most sensitive and fastest detectors for cocaine and offers near-continuous analyte measurement. KW - Drug search KW - Customs KW - Confiscation KW - Border surveillance KW - Narcotics KW - International drug trade KW - Drug trafficking KW - Illicit drug KW - Immunosensor KW - Antibodies KW - Detection PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-529951 DO - https://doi.org/10.20944/preprints202107.0521.v1 SN - 2310-287X N1 - Geburtsname von Ponader, Marco: Wilke, M. - Birth name of Ponader, Marco: Wilke, M. SP - 1 PB - MDPI CY - Basel AN - OPUS4-52995 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Ponader, Marco A1 - Röder, Bettina A1 - Paul, Martin A1 - Weller, Michael G. T1 - Sintered glass monoliths as new supports for affinity columns N2 - A novel stationary phase for affinity separations is presented. This material is based on sintered borosilicate glass readily available as semi-finished filter plates with defined porosity and surface area. The material shows fast binding kinetics and excellent long-term stability under real application conditions due to lacking macropores and high mechanical rigidity. The glass surface can be easily modified with standard organosilane chemistry to immobilize selective binders or other molecules used for biointeraction. In this paper, the manufacturing of the columns and their respective column holders by 3D printing is shown in detail. The model system protein A/IgG was chosen as an example to examine the properties of such monolithic columns under realistic application conditions. Several specifications, such as (dynamic) IgG capacity, pressure stability, long-term performance, productivity, non-specific binding, and peak shape, are presented. It could be shown that due to the very high separation speed, 250 mg antibody per hour and column can be collected, which surpasses the productivity of most standard columns of the same size. The total IgG capacity of the shown columns is around 4 mg (5.5 mg/mL), which is sufficient for most tasks in research laboratories. The cycle time of an IgG separation can be less than 1 minute. Due to the glass material's excellent pressure resistance, these columns are compatible with standard HPLC systems. This is usually not the case with standard affinity columns, limited to manual use or application in low-pressure systems. The use of a standard HPLC system also improves the ability for automation, which enables the purification of hundreds of cell supernatants in one day. The sharp peak shape of the elution leads to an enrichment effect, which might increase the concentration of IgG by a factor of 3. The final concentration of IgG can be around 7.5 mg/mL without the need for an additional nanofiltration step. The purity of the IgG was > 95% in one step and nearly 99% with a second polishing run. KW - Affinity Chromatography KW - Glass KW - Purification KW - Antibodies KW - Solid support KW - HPLC KW - FPLC KW - Separation PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-529117 DO - https://doi.org/10.20944/preprints202103.0298.v1 SN - 2310-287X N1 - Geburtsname von Ponader, Marco: Wilke, M. - Birth name of Ponader, Marco: Wilke, M. SP - 1 EP - 17 PB - MDPI CY - Basel AN - OPUS4-52911 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -