TY - JOUR A1 - Döring, Sarah A1 - Tscheuschner, Georg A1 - Flemig, Sabine A1 - Weller, Michael G. A1 - Konthur, Zoltán T1 - Cost-Effective Method for Full-Length Sequencing of Monoclonal Antibodies from Hybridoma Cells N2 - Background: Monoclonal antibodies play an important role in therapeutic and analytical applications. For recombinant expression, the coding sequences of the variable regions of the heavy and light chains are required. In addition, cloning antibody sequences, including constant regions, reduces the impact of hybridoma cell loss and ensures preservation of the naturally occurring full antibody sequence. Method: We combined amplification of IgG antibody variable regions from hybridoma mRNA with an advanced method for fulllength cloning of monoclonal antibodies in a simple two-step workflow. Following Sanger sequencing and evaluation of consensus sequences, the best matching variable, diversity, and joining (V-(D-)J) gene segments were identified according to identity scores from IgBLAST reference sequences. Simultaneously, the mouse IgG subclass was determined at the DNA level based on isotype-specific sequence patterns in the CH1 domain. Knowing the DNA sequence of V-(D-)J recombination responsible for the complementary determining region 3 (CDR 3), variable region-specific primers were designed and used to amplify the corresponding antibody constant regions. Results: To verify the approach, we applied it to the hybridoma clone BAM-CCMV-29-81 and obtained identical full-length antibody sequences as with RNA Illumina sequencing. Further validation at the protein level using an established MALDI-TOF MS-fingerprinting protocol showed that five out of six genetically encoded CDR domains of the monoclonal antibody BAM-CCMV-29-81 could be efficiently correlated. Conclusion: This simple, streamlined method enables the cost-effective determination of the full-length sequence of monoclonal antibodies from hybridoma cell lines, with the added benefit of obtaining the DNA sequence of the antibody ready for recombinant expression. KW - Full-length antibody sequencing KW - Hybridoma cell loss KW - MALDI-TOF MS KW - Immunoglobulin isotyping KW - RNA Illumina sequencing PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-639599 DO - https://doi.org/10.3390/antib14030072 SN - 2073-4468 VL - 14 IS - 3 SP - 1 EP - 17 PB - MDPI CY - Basel AN - OPUS4-63959 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Arakawa, Akihiro A1 - Jakubowski, Norbert A1 - Koellensperger, G. A1 - Theiner, S. A1 - Schweikert, A. A1 - Flemig, Sabine A1 - Iwahata, D. A1 - Traub, Heike A1 - Hirata, T. T1 - Imaging of Ag NP transport through collagen-rich microstructures in fibroblast multicellular spheroids by high-resolution laser ablation inductively coupled plasma time-of-flight mass spectrometry N2 - We investigated the penetration of silver nanoparticles (Ag NPs) into a three-dimensional in vitro tissue analog using NPs with various sizes and surface coatings, and with different incubation times. A high-Resolution laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) time-of-flight (TOF) instrument was applied for imaging the distributions of elements in thin sample sections (20 μm thick). A fibroblast multicellular spheroid (MCS) was selected as the model system and cultured for more than 8 days to produce a natural barrier formed by the extracellular matrix containing collagen. The MCS was then exposed for up to 48 h to one of four types of Ag NPs (∅ 5 nm citrate coated, ∅ 20 nm citrate coated, ∅ 20 nm polyvinylpyrrolidone coated, and ∅ 50 nm citrate coated). Imaging showed that the penetration pathway was strongly related to steric networks formed by collagen fibrils, and Ag NPs with a hydrodynamic diameter of more than 41 nm were completely trapped in an outer rim of the MCSs even after incubation for 48 h. In addition, we examined the impact of these NPs on essential elements (P, Fe, Cu, and Zn) in areas of Ag NP accumulation. We observed a linear increase at the sub-femtogram level in the total concentration of Cu (fg per pixel) in samples treated with small or large Ag NPs (∅ 5 nm or ∅ 50 nm) for 48 h. KW - Nanoparticle KW - Laser ablation KW - ICP-MS KW - Imaging KW - Cell PY - 2019 DO - https://doi.org/10.1039/c9an00856j SN - 0003-2654 VL - 144 IS - 16 SP - 4935 EP - 4942 PB - Royal Society of Chemistry RSC CY - Cambridge AN - OPUS4-48531 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Riedel, Soraya A1 - Hülagü, Deniz A1 - Bennet, Francesca A1 - Carl, Peter A1 - Flemig, Sabine A1 - Schmid, Thomas A1 - Schenk, J. A. A1 - Hodoroaba, Vasile-Dan A1 - Schneider, Rudolf T1 - Electrochemical Immunomagnetic Ochratoxin A Sensing: Steps Forward in the Application of 3,3’,5,5’- Tetramethylbenzidine in Amperometric Assays N2 - Electrochemical methods offer great promise in meeting the demand for user-friendly on-site devices for Monitoring important parameters. The food industry often runs own lab procedures, for example, for mycotoxin analysis, but it is a major goal to simplify analysis, linking analytical methods with smart technologies. Enzyme-linked immunosorbent assays, with photometric detection of 3,3’,5,5’-tetramethylbenzidine (TMB),form a good basis for sensitive detection. To provide a straightforward approach for the miniaturization of the detectionstep, we have studied the pitfalls of the electrochemical TMB detection. By cyclic voltammetry it was found that the TMB electrochemistry is strongly dependent on the pH and the electrode material. A stable electrode response to TMB could be achieved at pH 1 on gold electrodes. We created a smartphonebased, electrochemical, immunomagnetic assay for the detection of ochratoxin A in real samples, providing a solid basis forsensing of further analytes. KW - Ochratoxin A KW - Amperometry KW - Cyclic voltammetry KW - Electrochemistry KW - Immunoassay PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-530421 DO - https://doi.org/10.1002/celc.202100446 N1 - Geburtsname von Riedel, Soraya: Höfs, S. - Birth name of Riedel, Soraya: Höfs, S. VL - 8 IS - 13 SP - 2597 EP - 2606 AN - OPUS4-53042 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Devi, Sarita A1 - Riedel, Soraya A1 - Döring, Sarah A1 - Hiller, Lukas A1 - Kaliyaraj Selva Kumar, Archana A1 - Flemig, Sabine A1 - Singh, Chandan A1 - Konthur, Zoltán A1 - Hodoroaba, Vasile-Dan A1 - Schneider, Rudolf T1 - Antibodies Functionalized Magnetic Fe-Metal-Organic Framework Based Biosensor for Electrochemical Detection of Tetanus Neurotoxin N2 - This work presents a metal-organic framework (MOF)-integrated microfluidic flow-cell (MFC) based immunodetection of the tetanus toxoid (TT) using electrochemical technique for the first time. The magnetic property of Fe-MOF helped to hold them on the working electrode at detection zone of MFC surpassing the requirement of additional conjugation chemistry, whereas the conductive property was utilized to observe the change in signal efficiency in the presence of TT. The one-pot hydrothermal synthesis of a magnetic and conductive iron-based MOF (Fe-MOF) was performed using the Fe3 +/Fe2+ precursors as 1.2/1 mmol and dual ligands, i.e., tetrahydroxy-1,4-benzoquinone and 2-aminobenzene-1,4-dicarboxylic acid. The Fe-MOF was conjugated with L-phenylalanine (Fe-MOF/Phe) to increase its electric conductivity owing to the enhanced electron flow rate. The human monoclonal antibody SA13 against TT (anti-TT mAb) was conjugated on the Fe-MOF/Phe surface with the help of ethylenediamine (Fe-MOF/Phe/EDA/anti-TT mAb). The binding affinity of Fe-MOF/Phe/EDA/anti-TT mAb for the TT antigen was evaluated using cyclic voltammetry technique. The limit of detection of the Fe-MOF/Phe/EDA/anti-TT mAb-based biosensor for TT was 9.4 ng/ml in spiked buffer. This study shows the applicability of these Fe-MOFs in the detection of various other microbial toxins or other biomolecules. KW - Antikörper KW - Elektrochemischer Immunoassay KW - Molecular Organic Framework (MOF) KW - Microfluidics KW - Rekombinant PY - 2025 DO - https://doi.org/10.1016/j.snb.2025.137381 SN - 0925-4005 VL - 431 SP - 1 EP - 12 PB - Elsevier B.V. CY - Amsterdam AN - OPUS4-62673 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Arakawa, Akihiro A1 - Jakubowski, Norbert A1 - Koellensperger, G. A1 - Theiner, S. A1 - Schweikert, A. A1 - Flemig, Sabine A1 - Iwahata, D. A1 - Traub, Heike A1 - Hirata, T. T1 - Quantitative Imaging of Silver Nanoparticles and Essential Elements in Thin Sections of Fibroblast Multicellular Spheroids by High Resolution Laser Ablation Inductively Coupled Plasma Time-of-Flight Mass Spectrometry N2 - We applied high resolution laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with cellular spatial resolution for bioimaging of nanoparticles uptaken by fibroblast multicellular spheroids (MCS). This was used to quantitatively investigate interactions of silver nanoparticles (Ag NPs) and the distributions of intrinsic minerals and biologically relevant elements within thin sections of a fibroblast MCS as a three-dimensional in vitro tissue model. We designed matrix-matched calibration standards for this purpose and printed them using a noncontact piezo-driven array spotter with a Ag NP suspension and multielement standards. The limits of detection for Ag, Mg, P, K, Mn, Fe, Co, Cu, and Zn were at the femtogram (fg) level, which is sufficient to investigate intrinsic minerals in thin MCS sections (20 μm thick). After incubation for 48 h, Ag NPs were enriched in the outer rim of the MCS but not detected in the core. The localization of Ag NPs was inhomogeneous in the outer rim, and they were colocalized with a single-cell-like structure visualized by Fe distribution (pixel size of elemental images: 5 × 0.5 μm). The quantitative value for the total mass of Ag NPs in a thin section by the present method agreed with that obtained by ICP-sector field (SF)-MS with a liquid mode after acid digestion. KW - Laser ablation KW - ICP-MS KW - Imaging KW - Nanoparticle KW - Cell KW - Spheroid PY - 2019 UR - https://pubs.acs.org/doi/10.1021/acs.analchem.9b02239 DO - https://doi.org/10.1021/acs.analchem.9b02239 SN - 0003-2700 VL - 91 IS - 15 SP - 10197 EP - 10203 PB - American Chemical Society, ACS Publications CY - Washington D.C. AN - OPUS4-48719 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - INPR A1 - Devi, Sarita A1 - Riedel, Soraya A1 - Döring, Sarah A1 - Hiller, Lukas A1 - Flemig, Sabine A1 - Singh, Chandan A1 - Konthur, Zoltán A1 - Hodoroaba, Vasile-Dan A1 - Schneider, Rudolf T1 - Antibodies Functionalized Magnetic Fe-Metal-Organic Framework Based Biosensor for Electrochemical Detection of Tetanus NeuroToxin N2 - This work presents a MOF-integrated microfluidic flow-cell based immunodetection of the tetanus toxoid (TT) using electrochemical technique for the first time. Metal-organic frameworks (MOFs) are coordination polymers, and composed of a metal center and organic linkers. Several synthesis methods have been reported to achieve the desired properties in MOFs. In this work, we report the hydrothermal synthesis of a magnetic and conductive iron-based MOF (Fe-MOF) which was utilized to develop a biosensor in conjugation with the human monoclonal antibody SA13 against TT (anti-TT mAb) for the detection of the tetanus neurotoxin (TeNT). The one-pot synthesis of this magnetic and conductive Fe-MOF was performed in a hydrothermal reactor (108℃) using the Fe3+/Fe2+ precursors as 1.2/1 mmol and dual ligands, i.e., tetrahydroxy-1,4-benzoquinone and 2-aminobenzene-1,4-dicarboxylic acid. The Fe-MOF was characterized using XRD, DLS, FTIR, and electron microscopy. The Fe-MOF was further conjugated with L-phenylalanine (pH 8.6) to increase the electric conductivity of the Fe-MOF (Fe-MOF/Phe) on the screen-printed gold electrode as studied by cyclic voltammetry (CV). The anti-TT mAb was conjugated on the Fe-MOF/Phe surface with the help of ethylenediamine (Fe-MOF/Phe/EDA/anti-TT mAb) delivered good binding affinity for the TT antigen revealing the applicability of this biosensor for TT detection by CV. The limit of detection of the Fe-MOF/Phe/EDA/anti-TT mAb-based biosensor for TT was 9.4 ng/ml in spiked buffer. There is negligible cross-reactivity in the presence of bovine serum albumin. This study shows the applicability of magnetic MOFs in the detection of various other microbial toxins or other biomolecules. KW - Magnetic Metal-Organic Framework KW - Tetanus toxoid KW - L-phenylalanine KW - Antibody KW - Label-free detection KW - Electrochemical immunosensor PY - 2024 DO - https://doi.org/10.2139/ssrn.4935745 SP - 1 EP - 48 PB - Elsevier Inc. AN - OPUS4-62196 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Arakawa, Akihiro A1 - Jakubowski, Norbert A1 - Flemig, Sabine A1 - Koellensperger, G. A1 - Rusz, M. A1 - Iwahata, D. A1 - Traub, Heike A1 - Hirata, T. T1 - High-resolution laser ablation inductively coupled plasma mass spectrometry used to study transport of metallic nanoparticles through collagen-rich microstructures in fibroblast multicellular spheroids N2 - We have efficiently produced collagen-rich microstructures in fibroblast multicellular spheroids (MCSs) as a three-dimensional in vitro tissue analog to investigate silver (Ag) nanoparticle (NP) penetration. The MCS production was examined by changing the seeding cell number (500 to 40,000 cells) and the growth period (1 to 10 days). MCSs were incubated with Ag NP suspensions with a concentration of 5 μg/mL for 24 h. For this study, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to visualize Ag NP localization quantitatively. Thin sections of MCSs were analyzed by LA-ICP-MS with a laser spot size of 8 μm to image distributions of 109Ag, 31P, 63Cu, 66Zn, and 79Br. A calibration using a NP suspension was applied to convert the measured Ag intensity into the number of NPs present. The determined numbers of NPs ranged from 30 to 7200 particles in an outer rim of MCS. The particle distribution was clearly correlated with the presence of 31P and 66Zn and was localized in the outer rim of proliferating cells with a width that was equal to about twice the diameter of single cells. Moreover, abundant collagens were found in the outer rim of MCSs. For only the highest seeding cell number, NPs were completely captured at the outer rim, in a natural barrier reducing particle transport, whereas Eosin (79Br) used as a probe of small molecules penetrated into the core of MCSs already after 1 min of exposure. KW - Laser ablation KW - ICP-MS KW - Nanoparticle KW - Cell KW - Speroid PY - 2019 DO - https://doi.org/10.1007/s00216-019-01827-w SN - 1618-2642 SN - 1618-2650 VL - 411 IS - 16 SP - 3497 EP - 3506 PB - Springer CY - Berlin, Heidelberg AN - OPUS4-47900 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 - 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 -