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

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  • Sakaguchi, Atsumi
    Fac Engn, Lab Mol Biol, Yokohama National University Fac Engn, Lab Mol Biol, Open Facil Ctr, Biomat Anal Div, Tokyo Institute of Technology
  • Tanaka, Yoichiro
    Fac Engn, Lab Mol Biol, Open Facil Ctr, Biomat Anal Div, Instrumental Anal Ctr, Yokohama National University
  • Shoji, Eiki
    Fac Engn, Lab Mol Biol, Yokohama National University
  • Takeshima, Teppei
    Fac Engn, Lab Mol Biol, Open Facil Ctr, Biomat Anal Div, Instrumental Anal Ctr, Dept Urol & Renal Transplantat, Med Ctr, Yokohama City University
  • Sakamaki, Rina
    Fac Engn, Lab Mol Biol, Open Facil Ctr, Biomat Anal Div, Instrumental Anal Ctr, Dept Urol & Renal Transplantat, Med Ctr, Biosci Div, Tosoh Corp
  • Matsuba, Takao
    Fac Engn, Lab Mol Biol, Open Facil Ctr, Biomat Anal Div, Instrumental Anal Ctr, Dept Urol & Renal Transplantat, Med Ctr, Biosci Div, Tosoh Corp
  • Kurihara, Yasuyuki
    Fac Engn, Lab Mol Biol, Yokohama National University

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BackgroundMonoclonal 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.ResultsIn 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.ConclusionsThe 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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