Optimization of Electrofusion Parameters for Producing Hybridomas Synthesizing Human Monoclonal Antibodies

Силкина, М. В.; Карцева, А. С.; Ryabko, A. K.; Marin, M. A.; Romanenko, Yana O.; Kalmantaeva, O. V.; Khlyntseva, A. E.; Shemyakin, I. G.; Dyatlov, I. A.; Фирстова, В. В.

doi:10.1134/s0003683822090095

Cited by 2 publications

(4 citation statements)

References 23 publications

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“…Several studies have reported that the number of specific plasmablasts increases in the blood on day 7 or 8 after vaccination or infection [ 9 , 10 , 11 ]. To increase the number of produced hybridomas, we used electrofusion, whose efficiency is an order of magnitude higher than that of fusion with PEG [ 8 ]. The use of the K6H6/B5 cell line as partner cells reduced the risk of subsequent segregation of human chromosomes from hybridomas [ 12 ].…”

Section: Discussionmentioning

confidence: 99%

“…On day 7 after vaccination, peripheral blood was collected and the B lymphocyte fraction was isolated using a RossetteSep™ Human B Cell Enrichment Cocktail kit (Stemcell technologies, Canada), in accordance with the manufacturer’s instructions. The isolated B lymphocytes were electrofused with the K6H6/B5 myeloma cell line (ATCC® CRL1823™) using an ECM 2001 device (BTX Harvard Apparatus, USA), according to a previously published procedure [ 8 ].…”

Section: Methodsmentioning

confidence: 99%

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Characterization of the C6D7-RBD Human Monoclonal Antibody Specific to the SARS-CoV-2 S Protein Receptor-Binding Domain

et al. 2023

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The new coronavirus infection COVID-19 is an acute viral disease that affects primarily the upper respiratory tract. The etiological agent of COVID-19 is the SARS-CoV-2 RNA virus (Coronaviridae family, Betacoronavirus genus, Sarbecovirus subgenus). We have developed a high-affinity human monoclonal antibody, called C6D7-RBD, which is specific to the S protein receptor-binding domain (RBD) from the SARS-CoV-2 Wuhan-Hu-1 strain and exhibits virus-neutralizing activity in a test with recombinant antigens: angiotensin-converting enzyme 2 (ACE2) and RBD.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Characterization of the C6D7-RBD Human Monoclonal Antibody Specific to the SARS-CoV-2 S Protein Receptor-Binding Domain

et al. 2023

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“…Another limiting factor in the production of human hybridoma is the efficiency of fusion, which can be increased by choosing the optimal conditions and parameters. 36 …”

Section: Discussionmentioning

confidence: 99%

“…Previously, we have shown that the use of the K6H6/B5 line for hybridization with human B cells makes it possible to obtain a stable producer of huMAbs. 36 …”

Section: Introductionmentioning

confidence: 99%

New approach to generating of human monoclonal antibodies specific to the proteolytic domain of botulinum neurotoxin A

Silkina,

Kartseva,

Riabko

et al. 2023

Bioimpacts

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Introduction: Botulinum neurotoxins (BoNTs) cause botulism and are the most potent natural toxins known. Immunotherapy with neutralizing monoclonal antibodies (MAbs) is considered to be the most effective immediate response to BoNT exposure. Hybridoma technology remains the preferred method for producing MAbs with naturally paired immunoglobulin genes and with preserved innate functions of immune cells. The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against BoNTs. In an effort to develop novel BoNT type A (BoNT/A) immunotherapeutics, sorted by flow cytometry plasmablasts and activated memory B cells from a donor repeatedly injected with BoNT/A for aesthetic botulinum therapy could be used due to obtain hybridomas producing native antibodies. Methods: Plasmablasts and activated memory B-cells were isolated from whole blood collected 7 days after BoNT/A injection and sorted by flow cytometry. The sorted cells were then electrofused with the K6H6/B5 cell line, resulting in a producer of native human monoclonal antibodies (huMAbs). The 3 antibodies obtained were then purified by affinity chromatography, analyzed for binding by Western blot assay and neutralization by FRET assay. Results: We have succeeded in creating 3 hybridomas that secrete huMAbs specific to native BoNT/A and the proteolytic domain (LC) of BoNT/A. The 1B9 antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusion: The use activated plasmablasts and memory B-cells isolated at the peak of the immune response (at day 7 of immunogenesis) that have not yet completed the terminal stage of differentiation but have undergone somatic hypermutation for hybridization allows us to obtain specific huMAbs even when the immune response of the donor is weak (with low levels of specific antibodies and specific B-cells in blood). A BoNT/A LC-specific antibody is capable of effectively inhibiting BoNT/A by mechanisms not previously associated with antibodies that neutralize BoNT. Antibodies specific to BoNT LC can be valuable components of a mixture of antibodies against BoNT exposure.

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Optimization of Electrofusion Parameters for Producing Hybridomas Synthesizing Human Monoclonal Antibodies

Cited by 2 publications

References 23 publications

Characterization of the C6D7-RBD Human Monoclonal Antibody Specific to the SARS-CoV-2 S Protein Receptor-Binding Domain

Characterization of the C6D7-RBD Human Monoclonal Antibody Specific to the SARS-CoV-2 S Protein Receptor-Binding Domain

New approach to generating of human monoclonal antibodies specific to the proteolytic domain of botulinum neurotoxin A

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