TY - GEN A1 - Göthel, Markus A1 - Listek, Martin A1 - Messerschmidt, Katrin A1 - Schlör, Anja A1 - Hönow, Anja A1 - Hanack, Katja T1 - A New Workflow to Generate Monoclonal Antibodies against Microorganisms T2 - Mathematisch-Naturwissenschaftliche Reihe N2 - Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1174 KW - monoclonal antibody KW - antibody producing cell selection KW - hybridoma KW - epitope prediction Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-523341 SN - 1866-8372 IS - 20 ER - TY - JOUR A1 - Göthel, Markus A1 - Listek, Martin A1 - Messerschmidt, Katrin A1 - Schlör, Anja A1 - Hönow, Anja A1 - Hanack, Katja T1 - A New Workflow to Generate Monoclonal Antibodies against Microorganisms JF - Applied Sciences N2 - Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms. KW - monoclonal antibody KW - antibody producing cell selection KW - hybridoma KW - epitope prediction Y1 - 2021 U6 - https://doi.org/10.3390/app11209359 SN - 1454-5101 VL - 11 IS - 20 PB - MDPI CY - Basel ER - TY - JOUR A1 - Tscheuschner, Georg A1 - Kaiser, Melanie N. A1 - Lisec, Jan A1 - Beslic, Denis A1 - Muth, Thilo A1 - Krüger, Maren A1 - Mages, Hans Werner A1 - Dorner, Brigitte G. A1 - Knospe, Julia A1 - Schenk, Jörg A. A1 - Sellrie, Frank A1 - Weller, Michael G. T1 - MALDI-TOF-MS-based identification of monoclonal murine Anti-SARS-CoV-2 antibodies within one hour JF - Antibodies 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 - SARS-CoV-2 antibody KW - reproducibility crisis KW - peptide mass KW - fingerprinting KW - monoclonal antibody KW - traceability KW - identity KW - antibody KW - identification KW - antibody light chain KW - MALDI-TOF-MS Y1 - 2022 U6 - https://doi.org/10.3390/antib11020027 SN - 2073-4468 VL - 11 IS - 2 PB - MDPI CY - Basel ER - TY - JOUR A1 - Engel, Robert A1 - Micheel, Burkhard A1 - Hanack, Katja T1 - Three-dimensional cell culture approach for in vitro immunization and the production of monoclonal antibodies JF - Biomedical materials : materials for tissue engineering and regenerative medicine N2 - The generation of monoclonal antibodies using an in vitro immunization approach is a promising alternative to conventional hybridoma technology. As recently published, the in vitro approach enables an antigen-specific activation of B lymphocytes within 10-12 d followed by immortalization and subsequent selection of hybridomas. This in vitro process can be further improved by using a three-dimensional surrounding to stabilize the complex microenvironment required for a successful immune reaction. In this study, the suitability of Geltrex as a material for the generation of monoclonal antigen-specific antibodies by in vitro immunization was analyzed. We could show that dendritic cells, B cells, and T cells were able to travel through and interact inside of the matrix, leading to the antigen-specific activation of T and B cells. For cell recovery and subsequent hybridoma technique the suitability of dispase and Corning cell recovery solution (CRS) was compared. In our experiments, the use of dispase resulted in a severe alteration of cell surface receptor expression patterns and significantly higher cell death, while we could not detect an adverse effect of Corning CRS. Finally, an easy approach for high-density cell culture was established by printing an alginate ring inside a cell culture vessel. The ring was filled with Geltrex, cells, and medium to ensure a sufficient supply during cultivation. Using this approach, we were able to generate monoclonal hybridomas that produce antigen-specific antibodies against ovalbumin and the SARS-CoV-2 nucleocapsid protein. KW - monoclonal antibody KW - hybridoma technology KW - in vitro immunization KW - 3D KW - cell culture KW - Geltrex Y1 - 2022 U6 - https://doi.org/10.1088/1748-605X/ac7b00 SN - 1748-6041 SN - 1748-605X VL - 17 IS - 5 PB - Inst. of Physics CY - London ER -