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

Abstract

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.

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

“…DNA sequencing con rmed this DNA sequence. The recombinant proteins were puri ed as previously described [15].…”

“…The hybridoma used in this study was prepared by immunizing cells with anti-His-EGFP, followed by cell fusion, aliquoting the hybridoma cells into tubes, and cryopreservation. Both our previous study [15] and the present study used cells that were stocked at the same time. After culturing, hybridoma cells were screened using the MIHS method.…”

“…After culturing, hybridoma cells were screened using the MIHS method. Brie y, 2 nmol of His-EGFP was added to 1 mL of 5 × 10 5 hybridoma cell culture medium, following which the cells were cultured at 37 ℃ and 5% CO 2 for 2 h. The cells were then washed three times with serum-free RPMI 1640, and EGFP-positive cells were seeded individually using a MoFlo Astrios cell sorter (Beckman Coulter Inc, CA), as described in a previous study [15].…”

“…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.…”

Monoclonal Antibody Discovery Based on Precise Selection of Single Transgenic Hybridomas with an On-Cell-Surface and Antigen-Specific Anchor

“…16 More importantly, chemical modifications on the hybridoma surface cannot prevent mAbs from binding to the nearby hybridomas that do not secrete them, resulting in a high rate of false positives. Recently, Martin et al developed a gentle and stable transgenic myeloma with a biological anchor that combined the truncated variant transmembrane domain of the epidermal growth factor receptor (EGFR) and a biotin acceptor peptide for antigen-and isotype-specific hybridoma screening, 17 and this appears to be a promising replacement for chemical anchors. However, this strategy has not been used in mAb production.…”