Advanced hybridoma technology for selective production of high-affinity monoclonal antibodies through B-cell receptors

Sakashita, Kento; Tsumoto, K.; Tomita, Masahiro

doi:10.1016/j.jim.2022.113384

Cited by 4 publications

(5 citation statements)

References 36 publications

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“…B-cell receptor (BCR), a type of transmembrane immunoglobulin, has recently been gaining increasing attention owing to its applicability as a tag for screening structure-recognizing antibodies [ 12 – 17 ]. Hybridomas not only secrete soluble antibodies but also express BCRs on their cell membranes.…”

Section: Introductionmentioning

confidence: 99%

“…There are two strategies (chemical and enzymatic) for introducing biotin into antigen proteins. In chemical methods, biotin is introduced into the amino, thiol, and aldehyde groups of amino acids, such as lysine and cysteine [ 16 , 17 , 20 ]. However, this approach is not suitable for antigen production because biotin can modify antigenicity.…”

Section: Introductionmentioning

confidence: 99%

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Rapid, simple, and effective strategy to produce monoclonal antibodies targeting protein structures using hybridoma technology

et al. 2023

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Background Monoclonal antibodies are essential in life science research and developing antibody drugs and test drugs. Various methods have been developed to obtain monoclonal antibodies, among which hybridoma technology continues to be widely used. However, developing a rapid and efficient method for obtaining conformation-specific antibodies using hybridoma technology remains challenging. We previously developed the membrane-type immunoglobulin-directed hybridoma screening (MIHS) method, which is a flow cytometry-based screening technique based on the interaction between the B-cell receptor expressed on the hybridoma cell surface and the antigen protein, to obtain conformation-specific antibodies. Results In this study, we proposed a streptavidin-anchored ELISA screening technology (SAST) as a secondary screening method that retains the advantages of the MIHS method. Anti-enhanced green fluorescent protein monoclonal antibodies were generated as a model experiment, and their structural recognition abilities were examined. Examination of the reaction profiles showed that all monoclonal antibodies obtained in this study recognize the conformational epitopes of the protein antigen. Furthermore, these monoclonal antibodies were classified into two groups: those with binding activities against partially denatured proteins and those with complete loss of binding activities. Next, when screening monoclonal antibodies by the MIHS method as the first screening, we found that monoclonal antibodies with stronger binding constants may be selected by double-staining for hybridomas with fluorescently labeled target antigens and fluorescently labeled B cell receptor antibodies. Conclusions The proposed two-step screening method, which incorporates MIHS and SAST, constitutes a rapid, simple, and effective strategy to obtain conformation-specific monoclonal antibodies generated through hybridoma technology. The novel monoclonal antibody screening strategy reported herein could accelerate the development of antibody drugs and antibody tests.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rapid, simple, and effective strategy to produce monoclonal antibodies targeting protein structures using hybridoma technology

et al. 2023

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“…[1][2][3][4] The production times for antibodies can be several months due to the time required for antibody-epitope pair identification and subsequent antibody production, and potential mutations can lead to further delays. [6][7][8] Therefore, for rapid response and screening, an alternative means of biorecognition with quicker production times but similar selectivity and detection limits is desired.…”

Section: Introductionmentioning

confidence: 99%

Rapid sub-nanomolar protein determination in serum using electropolymerized molecularly imprinted polymers (E-MIPs)

Stephen,

Dennison,

Holden

et al. 2023

Analyst

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“…Therefore, to obtain mAbs for drug testing and support the development of therapeutic drugs for emerging infectious diseases, a rapid, simple, and effective strategy to produce mAbs that recognize the native structure of a target protein is required. B-cell receptor (BCR), a type of transmembrane immunoglobulin, has recently been gaining increasing attention owing to its applicability as a tag for screening structure-recognizing antibodies [12,13,14,15,16,17]. Hybridomas not only secrete soluble antibodies but also express BCRs on their cell membranes.…”

Section: Introductionmentioning

confidence: 99%

“…There are two strategies (chemical and enzymatic) for introducing biotin into antigen proteins. In chemical methods, biotin is introduced into the amino, thiol, and aldehyde groups of amino acids, such as lysine and cysteine [16,17,20]. However, this approach is not suitable for antigen production because biotin can modify antigenicity.…”

Section: Introductionmentioning

confidence: 99%

Rapid, simple, and effective strategy to produce monoclonal antibodies targeting native protein structures using hybridoma technology

Sakaguchi

Tanaka

Shoji

et al. 2022

Preprint

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BACKGROUND: Monoclonal antibodies are essential in life science research and developing antibody drugs and test drugs. Various methods have been developed to obtain monoclonal antibodies, among which hybridoma technology continues to be widely used. However, developing a rapid and efficient method for obtaining conformation-specific antibodies using hybridoma technology remains challenging. We previously developed the membrane-type immunoglobulin-directed hybridoma screening (MIHS) method, which is a flow cytometry-based screening technique based on the interaction between the B-cell receptor expressed on the hybridoma cell surface and the antigen protein, to obtain conformation-specific antibodies. RESULTS: In this study, we proposed a streptavidin-anchored ELISA screening technology (SAST) as a secondary screening method that retains the advantages of the MIHS method. Anti-enhanced green fluorescent protein monoclonal antibodies were generated as a model experiment, and their structural recognition abilities were examined. Examination of the reaction profiles showed that all monoclonal antibodies obtained in this study recognize the native protein structure. Furthermore, these monoclonal antibodies were classified into two groups: those with binding activities against partially denatured proteins and those with complete loss of binding activities. Next, when screening monoclonal antibodies by the MIHS method as the first screening, we found that monoclonal antibodies with stronger binding constants may be selected by double-staining for hybridomas with fluorescently labeled target antigens and fluorescently labeled B cell receptor antibodies. CONCLUSIONS: The proposed two-step screening method, which incorporates MIHS and SAST, constitutes a rapid, simple, and effective strategy to obtain conformation-specific monoclonal antibodies generated through hybridoma technology. The novel monoclonal antibody screening strategy reported herein could accelerate the development of antibody drugs and antibody tests.

show abstract

Advanced hybridoma technology for selective production of high-affinity monoclonal antibodies through B-cell receptors

Cited by 4 publications

References 36 publications

Rapid, simple, and effective strategy to produce monoclonal antibodies targeting protein structures using hybridoma technology

Rapid, simple, and effective strategy to produce monoclonal antibodies targeting protein structures using hybridoma technology

Rapid sub-nanomolar protein determination in serum using electropolymerized molecularly imprinted polymers (E-MIPs)

Rapid, simple, and effective strategy to produce monoclonal antibodies targeting native protein structures using hybridoma technology

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