Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Holzlöhner, Pamela; Hanack, Katja

doi:10.3791/54832

Cited by 40 publications

(35 citation statements)

References 20 publications

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“…A PE-conjugated F(ab) 2 fragment of a donkey anti-mouse IgG (12-4012, eBioscience, UK) diluted in PBS containing 0.5% BSA and 2 mM EDTA was used as indicator antibody. 40 . Briefly, spleen lymphocytes were mixed in a ratio of 3 to 1 with transfected myeloma cells that bear the artificial cell surface fusion protein and were washed three times with fusion buffer (125 mM NaCl, 5 mM KCl, 4 mM CaCl 2 , 2.5 mM MgCl 2 , 5 mM Tris-HCl pH 7.4, 0.2 µm).…”

Section: Flow Cytometry Analysis Of Using the Artificial Receptor As mentioning

confidence: 99%

A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line

Listek

Hönow

Gossen

et al. 2020

Sci Rep

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the use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. to address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. for the design of the construct we combined the transmembrane domain of the eGf-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. the stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen-and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.

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Section: Flow Cytometry Analysis Of Using the Artificial Receptor As mentioning

confidence: 99%

A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line

Listek

Hönow

Gossen

et al. 2020

Sci Rep

Self Cite

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“…For coupling the synthesized antigen to carrier protein, several methods can be considered (Yokoyama et al, 2013;Greenfield, 2014;Ossipow and Fischer, 2014;Holzlöhner and Hanack, 2017), depending on the sequence of the peptide and the compatibility with the chemical synthesis of the Ub-peptide conjugates. Here we describe a general crosslinking protocol that uses glutaraldehyde for amine-to-amine coupling; the internal lysines on Ub provide several amines for efficient coupling to the carrier protein.…”

Section: Antigen Crosslinkingmentioning

confidence: 99%

“…The generation of monoclonal antibodies in mice involves multiple steps: (i) design and synthesis of non-hydrolyzable Ubpeptide conjugates for immunization; (ii) design and synthesis of extended native iso-peptide linked Ub-peptide conjugates for screening; (iii) immunization, and generation and screening of hybridomas; (iv) clone selection and antibody validation in native context (Figure 1). In this manuscript we focus on the steps that are specific for synthesis of ubiquitin-peptide conjugates for the generation of site-specific ubiquitin antibodies (steps i and ii), as detailed excellent general protocols for antibody development (step iii) and validation (step iv) have been described elsewhere (Egelhofer et al, 2010;Yokoyama et al, 2013;Greenfield, 2014;Ossipow and Fischer, 2014;Kungulovski et al, 2015;Marcon et al, 2015;Rothbart et al, 2015;Uhlen et al, 2016;Guillemette et al, 2017;Holzlöhner and Hanack, 2017;Edfors et al, 2018;Venkataraman et al, 2018;Weller, 2018;Marx, 2019). In addition, we discuss the rationale for the design of antigens used for immunization and screening.…”

Section: Introductionmentioning

confidence: 99%

Strategy for Development of Site-Specific Ubiquitin Antibodies

et al. 2020

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Protein ubiquitination is a key post-translational modification regulating a wide range of biological processes. Ubiquitination involves the covalent attachment of the small protein ubiquitin to a lysine of a protein substrate. In addition to its well-established role in protein degradation, protein ubiquitination plays a role in protein-protein interactions, DNA repair, transcriptional regulation, and other cellular functions. Understanding the mechanisms and functional relevance of ubiquitin as a signaling system requires the generation of antibodies or alternative reagents that specifically detect ubiquitin in a site-specific manner. However, in contrast to other post-translational modifications such as acetylation, phosphorylation, and methylation, the instability and size of ubiquitin−76 amino acids-complicate the preparation of suitable antigens and the generation antibodies detecting such site-specific modifications. As a result, the field of ubiquitin research has limited access to specific antibodies. This severely hampers progress in understanding the regulation and function of site-specific ubiquitination in many areas of biology, specifically in epigenetics and cancer. Therefore, there is a high demand for antibodies recognizing site-specific ubiquitin modifications. Here we describe a strategy for the development of site-specific ubiquitin antibodies. Based on a recently developed antibody against site-specific ubiquitination of histone H2B, we provide detailed protocols for chemical synthesis methods for antigen preparation and discuss considerations for screening and quality control experiments.

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“…The concentrations of purified ascites were determined by Bradford method. The class and subclass of mAbs were determined by immunoType ™ mouse monoclonal antibody isotyping kit (Thermo Fisher, Waltham, MA, USA, Cat#26178) [18][19][20]. Following this process and primary screen, we obtained monoclonal antibodies against ZEBOV GP or VP40, which summarized in Table 1.…”

Section: Design and Production Of Mabsmentioning

confidence: 99%

Development and Characterization of Neutralizing Antibodies Against Zaire Ebolavirus Glycoprotein and Protein 40

Weng

Shen

et al. 2018

Cell Physiol Biochem

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Background/Aims: Monoclonal antibodies (mAbs) are presently the most promising treatment against Ebola virus disease (EVD), and cocktail of two or more antibodies likely confers protection through complementary mechanisms. Zaire Ebolavirus (EBOV) glycoprotein (GP) and viral protein 40 (VP40) are targets for designing neutralizing antibodies. Currently, the antiviral therapeutics of mAb-cocktails are still limited solely to anti-GP antibodies，there is no Abs cocktail against Zaire EBOV GP and VP40, which both have important interactions with host cellular membrane. Methods: We used hybridoma technology to produce anti-Zaire EBOV GP mAb against GP receptor binding domain, and anti-Zaire EBOV VP40 mAbs against the N-terminal domain, the C-terminal domain, respectively; synthesized Zaire EBOV transcription and replication competent virus like particles (trVLPs), which model even all aspects of the EBOV life cycles in order to evaluate the anti-viral effect of mAbs. Then, we characterized the anti- Zaire EBOV trVLPs effect of anti-GP and VP40 mAbs in vitro by real time-PCR, immunofluorescence assay and western blot analysis. Results: Our results demonstrate that anti-GP or anti-VP40 mAbs effectively inhibit trVLPs replication. The cocktails of anti-GP and anti-VP40 mAbs, or between anti-VP40 mAbs, had synergistic anti-trVLPs effect. Meanwhile, the detailed DNA and amino acid sequences of the mAbs were checked. Conclusion: The study verifies neutralizing efficacy of anti-GP or anti-VP40 mAb, report promising cocktail of anti-GP and anti-VP40 mAb, or cocktail of two anti-VP40 mAbs. To our knowledge, this is the first account to report the important anti-viral effect of cocktails of anti-GP and anti-VP40 mAbs in vitro.

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Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Cited by 40 publications

References 20 publications

A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line

A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line

Strategy for Development of Site-Specific Ubiquitin Antibodies

Development and Characterization of Neutralizing Antibodies Against Zaire Ebolavirus Glycoprotein and Protein 40

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