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

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.

Rapid and reliable hybridoma screening method that is suitable for production of functional structure-recognizing monoclonal antibody.

Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer.

Coxfa4l3, a novel mitochondrial electron transport chain Complex 4 subunit protein, switches from Coxfa4 during spermatogenesis.

We identified Coxfa4l3, previously called C15orf48 or Nmes1, as a novel accessory protein of Complex IV of the mitochondrial electron transport chain (ETC). Amino acid sequence comparison, the intracellular localization and the protein expression data showed that the protein is the third isoform of Coxfa4 and the expression of Coxfa4 and Coxfa4l3 proteins during spermatogenesis showed a mutually exclusive pattern, implying that Coxfa4 replaces Coxfa4l3 in Complex IV after meiosis. These results may provide some insight into the unique mechanism of ATP production in late spermatogenesis.