Comprehensive method for producing high-affinity mouse monoclonal antibodies of various isotypes against (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten

Rin Yoshizato; Mariko Miura; Kiyomi Shitaoka; Yuri Matsuoka; Akifumi Higashiura; Akima Yamamoto; Yun Guo; Hitoshi Azuma; Yohei Kawano; Shouichi Ohga; Tomoharu Yasuda

doi:10.1016/j.heliyon.2024.e40837

Comprehensive method for producing high-affinity mouse monoclonal antibodies of various isotypes against (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten

Heliyon. 2024 Nov 29;10(23):e40837. doi: 10.1016/j.heliyon.2024.e40837. eCollection 2024 Dec 15.

Authors

Affiliations

¹ Department of Immunology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
² Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
³ Department of Virology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Abstract

Monoclonal antibody (mAb) technology has significantly contributed to basic research and clinical settings for various purposes, including protective and therapeutic drugs. However, a rapid and convenient method to generate high-affinity antigen-specific mAbs has not yet been reported. Here, we developed a rapid, easy, and low-cost protocol for antigen-specific mAb production from single memory B cells. Using this method, high-affinity IgG1 mAbs specific to the hapten 4-hydroxy-3-nitrophenylacetyl (NP) were established from NP-CGG immunized C57BL/6 mice within 6 days. Our mAb production system allows flexible switching of IgG1 to any other isotype with the same paratope, enabling the absolute quantification of antigen-specific serum antibody titers and affinity maturation. Additionally, we established a protocol for the production of IgM and IgA, retaining their functional pentamer and dimer structures. This method is also effective against human antigens and pathogens, making it a powerful tool for mAb development in both research and clinical settings.

Keywords: 4-Hydroxy-3-nitrophenylacetyl (NP); Affinity maturation; B cell receptor; IgA; IgE; IgG; IgM; J chain; Memory B cells; Monoclonal antibody; Secretory component; Single cell.