TY - JOUR A1 - Carvalho, José Joao A1 - Walter, Astrid A1 - Baermann, Yvonne A1 - Weller, Michael G. A1 - Panne, Ulrich A1 - Schenk, J.A. A1 - Schneider, Rudolf T1 - Non-invasive monitoring of immunization progress in mice via IgG from feces N2 - A non-invasive method to monitor the humoral immune response in mice after immunization is described. From fecal pellets of an individual mouse, a sufficient amount of active immunoglobulins or their fragments can be extracted to perform a regular examination of the status of the immune response by immunoassay. Hapten-specific antibodies from the feces of mice from three immunization trials showed very similar characteristics to those obtained from serum at a given date. Therefore, it can be suspected that some serum IgG enters the intestinal lumen and ends up in the feces, where they appear to be considerably stable. Hapten-specific IgAs were not found in the feces. Being able to analyze antibody titers in feces could be an interesting animal welfare refinement to standard practice that does not entail repeated blood sampling. KW - Immunization monitoring KW - IgG KW - Coproantibodies KW - Feces KW - Animal welfare KW - 3R concept PY - 2012 UR - http://iv.iiarjournals.org/content/26/1/63.full.pdf+html SN - 0258-851x VL - 26 IS - 1 SP - 63 EP - 70 PB - In vivo CY - Athens AN - OPUS4-25283 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR 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 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to 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 60 min 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 degrees 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. 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 - Reproducibility KW - Quality control KW - Traceability KW - Peptides KW - Peptide mass fingerprinting KW - Monoclonal antibody KW - Recombinant antibody KW - Identity KW - Antibody identification KW - Sequencing KW - Light chain KW - Mass spectrometry KW - Software KW - Open science KW - Library KW - COVID-19 KW - Corona virus KW - Sequence coverage KW - NIST-mAb 8671 KW - Reference material KW - RBD KW - Spike protein KW - Nucleocapsid KW - Cleavage KW - Tryptic digest KW - MALDI KW - DHAP KW - 2,5-dihydroxyacetophenone KW - Github KW - Zenodo KW - ABID PY - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-547347 VL - 11 IS - 2 SP - 1 EP - 22 PB - MDPI CY - Basel AN - OPUS4-54734 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 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-545182 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 - JOUR A1 - Dippong, Martin A1 - Carl, Peter A1 - Lenz, C. A1 - Schenk, J. A. A1 - Hoffmann, Katrin A1 - Schwaar, Timm A1 - Schneider, Rudolf A1 - Kuhne, Maren T1 - Hapten-specific single-cell selection of hybridoma clones by fluorescence-activated cell sorting for the generation of monoclonal antibodies N2 - The conventional hybridoma screening and subcloning process is generally considered to be one of the most critical steps in hapten-specific antibody production. It is time-consuming, monoclonality is not guaranteed, and the number of clones that can be screened is limited. Our approach employs a novel hapten-specific labeling technique of hybridoma cells. This allows for fluorescence-activated cell sorting (FACS) and single-cell deposition and thereby eliminates the above-mentioned problems. A two-step staining approach is used to detect antigen specificity and antibody expression: in order to detect antigen specificity, hybridoma cells are incubated with a hapten−horseradish peroxidase conjugate (hapten−HRP), which is subsequently incubated with a fluorophore-labeled polyclonal anti-peroxidase antibody (anti-HRP−Alexa Fluor 488). To characterize the expression of membrane-bound immunoglobulin G (IgG), a fluorophore-labeled anti-mouse IgG antibody (anti-IgG−Alexa Fluor 647) is used. Hundreds of labeled hybridoma cells producing monoclonal antibodies (mAbs) specific for a hapten were rapidly isolated and deposited from a fusion mixture as single-cell clones via FACS. Enzyme-linked immunosorbent assay (ELISA) measurements of the supernatants of the sorted hybridoma clones revealed that all hapten-specific hybridoma clones secrete antibodies against the target. There are significant improvements using this high-throughput technique for the generation of mAbs including increased yield of antibody-producing hybridoma clones, ensured monoclonality of sorted cells, and reduced development times. KW - Monoclonal antibodies KW - FACS KW - Hybridoma cells PY - 2017 U6 - https://doi.org/10.1021/acs.analchem.6b04569 SN - 0003-2700 SN - 1520-6882 VL - 89 IS - 7 SP - 4007 EP - 4012 PB - ACS Publications AN - OPUS4-40320 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Singh, Chandan A1 - Riedel, Soraya A1 - Konthur, Zoltán A1 - Hodoroaba, Vasile-Dan A1 - Radnik, Jörg A1 - Schenk, J. A. A1 - Schneider, Rudolf T1 - Functionalized Ti3C2Tx nanosheets based biosensor for point-of-care detection of SARS-CoV‑2 antigen N2 - MXenes are considered a promising class of two-dimensional materials with extraordinary physical and electrochemical properties. Distinguished features like high specific surface area and outstanding electrical conductivity make them suitable for electrochemical biosensing applications. Here, we report the development of a biosensor involving the functionalized MXene−titanium carbide nanosheets (Ti3C2Tx-NS) and monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein (anti-SARS-CoV-2 mAb) to design a point-of-care device for detection of the SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 NP) antigen. Few-layered titanium carbide nanosheets (denoted as FL-Ti3C2Tx-NS) have been synthesized using a single-step etching and delamination method and characterized using optical and electron microscopy techniques revealing the suitability for immunosensing applications. Binding studies revealed the excellent affinity between the biosensor and the SARS-CoV-2 NP. Electrochemical detection of SARS-CoV-2 NP is performed using differential pulse voltammetry and read by a smartphone-based user interface. The proposed FL-Ti3C2Tx-NS based biosensor offers the detection of SARS-CoV-2 NP with a limit of detection of 0.91 nM in a wide detection range in spiked saliva samples. Additionally, there is no cross-reactivity in the presence of potential interferants like SARS-CoV-2 spike glycoprotein and bovine serum albumin. These findings demonstrate the potential of MXenes in developing a rapid and reliable tool for SARS-CoV-2 NP detection. While we report the biosensing of SARS-CoV-2 NP, our system also paves the way for the detection of other SARS-CoV-2 antigens like spike protein or other biomolecules based on antigen−antibody interactions. KW - Antigen testing KW - Few-layered titanium carbide nanosheets KW - SARS-CoV-2 nucleocapsid protein KW - Label-free detection KW - Electrochemical immunosensor PY - 2023 U6 - https://doi.org/10.1021/acsaenm.2c00118 SN - 2771-9545 N1 - Geburtsname von Riedel, Soraya: Höfs, S. - Birth name of Riedel, Soraya: Höfs, S. VL - 1 IS - 1 SP - 495 EP - 507 PB - American Chemical Society CY - Washington, DC AN - OPUS4-56931 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 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-530421 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 - Kuhne, Maren A1 - Dippong, Martin A1 - Flemig, Sabine A1 - Hoffmann, Katrin A1 - Petsch, K. A1 - Schenk, J.A. A1 - Kunte, Hans-Jörg A1 - Schneider, Rudolf T1 - Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA N2 - A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. KW - Immunization KW - Hapten KW - Monoclonal antibodies KW - Hybridoma KW - Flow cytometry KW - ELISA KW - Estradiol KW - Estrone KW - Digoxigenin KW - Zearalenone KW - Aflatoxin KW - CLSM PY - 2014 U6 - https://doi.org/10.1016/j.jim.2014.07.004 SN - 0022-1759 SN - 1872-7905 VL - 413 SP - 45 EP - 56 PB - Elsevier B.V. CY - Amsterdam AN - OPUS4-32322 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -