TY - JOUR A1 - Michelchen, Sophia A1 - Micheel, Burkhard A1 - Hanack, Katja T1 - In vitro immunization approach to generate specific murine monoclonal IgG antibodies JF - Journal of immunological methods : JIM N2 - Generating a monoclonal antibody to date is a time intense process that requires immunization of laboratory animals. The transfer of the humoral immune response into in vitro settings enables a shortening of this process and circumvents the necessity of in vivo immunization. However, to orchestrate the complex interplay of dendritic cells, T and B lymphocytes in vitro is very challenging. We therefore aimed for a simplified approach focusing on the protagonist of antibody production: the B lymphocyte. We activated purified murine B lymphocytes alone in vitro by using combinations of antigen and stimuli. We were able to induce a specific antibody response within ten days of culture against a viral coat protein as model antigen. Antibodies were of both IgM and IgG subclass. The stimulated B lymphocytes were transformed into permanently antibody-producing hybridomas by cell fusion technology. We furthermore used this method to induce a specific antibody response against L. pneumophila in vitro. We thus established a useful and effective in vitro protocol to generate monoclonal antibodies. By overcoming the necessity of in vivo immunization this protocol may be the first step towards a universal strategy to generate antibodies from various species. KW - Monoclonal antibody KW - Hybridoma technology KW - In vitro immunization KW - B cell activation Y1 - 2021 U6 - https://doi.org/10.1016/j.jim.2021.113149 SN - 0022-1759 SN - 1872-7905 VL - 499 PB - Elsevier CY - Amsterdam 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 - TY - JOUR A1 - Holzlöhner, Pamela A1 - Hanack, Katja T1 - Generation of murine monoclonal antibodies by hybridoma technology JF - JoVE : Video journal N2 - Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system. KW - Immunology KW - Issue 119 KW - monoclonal antibodies KW - hybridoma technology KW - myeloma cells KW - B lymphocytes KW - antigen KW - immunconjugate Y1 - 2017 U6 - https://doi.org/10.3791/54832 SN - 1940-087X IS - 119 PB - JoVE CY - Cambridge ER - TY - GEN A1 - Schlör, Anja A1 - Hirschberg, Stefan A1 - Ben Amor, Ghada A1 - Meister, Toni Luise A1 - Arora, Prerna A1 - Pöhlmann, Stefan A1 - Hoffmann, Markus A1 - Pfänder, Stephanie A1 - Eddin, Omar Kamal A1 - Kamhieh-Milz, Julian A1 - Hanack, Katja T1 - SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Introduction: The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications. Objectives: In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful tools for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity. Methods: Five antibody candidates were selected out of a naïve camelid library by phage display and expressed as full length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron). Results: All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50). Conclusion: Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1280 KW - camelid heavy-chain-only antibodies KW - single domain antibodies KW - nanobodies KW - SARS-CoV-2 KW - neutralization KW - Omicron Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-570124 SN - 1866-8372 IS - 1280 ER - TY - JOUR A1 - Schlör, Anja A1 - Hirschberg, Stefan A1 - Ben Amor, Ghada A1 - Meister, Toni Luise A1 - Arora, Prerna A1 - Pöhlmann, Stefan A1 - Hoffmann, Markus A1 - Pfänder, Stephanie A1 - Eddin, Omar Kamal A1 - Kamhieh-Milz, Julian A1 - Hanack, Katja T1 - SARS-CoV-2 neutralizing camelid heavy-chain-only antibodies as powerful tools for diagnostic and therapeutic applications JF - Frontiers in Immunology N2 - Introduction: The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications. Objectives: In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful tools for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity. Methods: Five antibody candidates were selected out of a naïve camelid library by phage display and expressed as full length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron). Results: All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50). Conclusion: Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy. KW - camelid heavy-chain-only antibodies KW - single domain antibodies KW - nanobodies KW - SARS-CoV-2 KW - neutralization KW - Omicron Y1 - 2022 U6 - https://doi.org/10.3389/fimmu.2022.930975 SN - 1664-3224 SP - 1 EP - 14 PB - Frontiers Media SA CY - Lausanne, Schweiz ER - TY - GEN A1 - Czarnecka, Malgorzata A1 - Weichelt, Ulrike A1 - Rödiger, Stefan A1 - Hanack, Katja T1 - Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1274 KW - antibody KW - camelid antibody KW - heavy-chain-only antibody KW - miRNA KW - nucleic acids KW - novel biomarkers Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-569142 SN - 1866-8372 SP - 1 EP - 18 ER - TY - JOUR A1 - Czarnecka, Malgorzata A1 - Weichelt, Ulrike A1 - Rödiger, Stefan A1 - Hanack, Katja T1 - Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA JF - International Journal of Molecular Sciences N2 - The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures. KW - antibody KW - camelid antibody KW - heavy-chain-only antibody KW - miRNA KW - nucleic acids KW - novel biomarkers Y1 - 2022 U6 - https://doi.org/10.3390/ijms23116275 SN - 1422-0067 VL - 23 SP - 1 EP - 18 PB - MDPI CY - Basel, Schweiz ET - 11 ER - TY - JOUR A1 - Fudickar, Werner A1 - Roder, Phillip A1 - Listek, Martin A1 - Hanack, Katja A1 - Linker, Torsten T1 - Pyridinium alkynylanthracenes as sensitizers for photodynamic therapy JF - Photochemistry and photobiology N2 - Photodynamic therapy (PDT) is a mild but effective method to treat certain types of cancer upon irradiation with visible light. Here, three isomeric methylpyridinium alkynylanthracenes 1op were evaluated as sensitizers for PDT. Upon irradiation with blue or green light, all three compounds show the ability to initiate strand breaks of plasmid DNA. The mayor species responsible for cleavage is singlet oxygen (O-1(2)) as confirmed by scavenging reagents. Only isomers 1m and 1p can be incorporated into HeLa cells, whereas isomer 1o cannot permeate through the membrane. While isomer 1m targets the cell nucleus, isomer 1p assembles in the cellular cytoplasm and impacts the cellular integrity. This is in accordance with a moderate toxicity of 1p in the dark, whereas 1m exhibits no dark toxicity. Both isomers are suitable as PDT reagents, with a CC50 of 3 mu m and 75 nm, for 1p and 1m, respectively. Thus, derivative 1m, which can be easily synthesized, becomes an interesting candidate for cancer therapy. Y1 - 2021 U6 - https://doi.org/10.1111/php.13554 SN - 0031-8655 SN - 1751-1097 VL - 98 IS - 1 SP - 193 EP - 201 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Lütkecosmann, Steffi A1 - Warsinke, Axel A1 - Tschöpe, Winfried A1 - Eichler, Rüdiger A1 - Hanack, Katja T1 - A novel monoclonal antibody suitable for the detection of leukotriene B4 JF - Biochemical and biophysical research communications N2 - Leukotriene B4 as an inflammatory mediator is an important biomarker for different respiratory diseases like asthma, chronic obstructive pulmonary disease or cystic lung fibrosis. Therefore the detection of LTB4 is helpful in the diagnosis of these pulmonary diseases. However, until now its determination in exhaled breath condensates suffers from problems of accuracy. Reasons for that could be improper sample collection and preparation methods of condensates and the lack of consistently assay specificity and reproducibility of the used immunoassay detection system. In this study we describe the development and the characterization of a specific monoclonal antibody (S27BC6) against LTB4, its use as molecular recognition element for the development of an enzyme-linked immunoassay to detect LTB4 and discuss possible future diagnostic applications. KW - Leukotriene B4 KW - Monoclonal antibody KW - Immunosensor KW - Chronic obstructive pulmonary disease (COPD) KW - Hapten Y1 - 2017 U6 - https://doi.org/10.1016/j.bbrc.2016.11.157 SN - 0006-291X SN - 1090-2104 VL - 482 IS - 4 SP - 1054 EP - 1059 PB - Elsevier CY - San Diego ER - TY - JOUR A1 - Holzlöhner, Pamela A1 - Butze, Monique A1 - Maier, Natalia A1 - Hebel, Nicole A1 - Schliebs, Erik A1 - Micheel, Burkhard A1 - Fuener, Jonas A1 - Heidicke, Gabriele A1 - Hanack, Katja T1 - Generation of murine monoclonal antibodies with specificity against conventional camelid IgG1 and heavy-chain only IgG2/3 JF - Veterinary Immunology and Immunopathology N2 - Camelids possess antibodies with a conventional four-chain structure consisting of two heavy and two light chains (of subclass IgG1) but further they also generate heavy-chain only antibodies (of subclass IgG2 and 3) which are fully functional in antigen binding. In this study subclass-specific murine monoclonal antibodies specific to conventional camelid IgG1 and heavy-chain only IgG2/3 were generated and validated for the use as potent secondary detection reagents. The monoclonal antibodies are able to differentiate between all camelid IgGs, conventional four-chain camelid antibodies (of subclass IgG1) and exclusively heavy chain-only antibodies (of subclasses IgG2 and IgG3). Further these antibodies were used to detect specific immune responses after vaccination of Camelids against bovine corona- and rotavirus strains and different E.coli. and Clostridia - antigens and to identify Erysipelothrix rhusiopathiae infected animals within a herd. The described antibodies are suitable as new secondary agents for the detection of different camelid subclasses and the validation of camelid immune reactions. KW - Camelid antibodies KW - Heavy-chain only antibodies KW - Monoclonal antibodies KW - Secondary antibodies KW - Vaccination KW - Disease monitoring Y1 - 2018 U6 - https://doi.org/10.1016/j.vetimm.2018.01.006 SN - 0165-2427 SN - 1873-2534 VL - 197 SP - 1 EP - 6 PB - Elsevier CY - Amsterdam ER -