Sakaguchi Atsumi, Tanaka Yoichiro, Shoji Eiki, Takeshima Teppei, Sakamaki Rina, Matsuba Takao, Kurihara Yasuyuki
Laboratory of Molecular Biology, Faculty of Engineering, Yokohama National University, 79-5, Tokiwadai, Hodogaya, Yokohama, Kanagawa, 240-8501, Japan.
Biomaterials Analysis Division, Open Facility Center, Tokyo Institute of Technology, Midori, Yokohama, Kanagawa, Japan.
J Biol Eng. 2023 Mar 30;17(1):24. doi: 10.1186/s13036-023-00345-9.
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.
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.
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.
单克隆抗体在生命科学研究、抗体药物研发及检测药物方面至关重要。已开发出多种获取单克隆抗体的方法，其中杂交瘤技术仍被广泛应用。然而，利用杂交瘤技术开发一种快速高效获取构象特异性抗体的方法仍具有挑战性。我们之前开发了膜型免疫球蛋白导向的杂交瘤筛选（MIHS）方法，这是一种基于流式细胞术的筛选技术，基于杂交瘤细胞表面表达的B细胞受体与抗原蛋白之间的相互作用来获取构象特异性抗体。
在本研究中，我们提出了链霉亲和素锚定ELISA筛选技术（SAST）作为二级筛选方法，该方法保留了MIHS方法的优点。以抗增强型绿色荧光蛋白单克隆抗体作为模型实验，并检测其结构识别能力。对反应图谱的检测表明，本研究中获得的所有单克隆抗体均识别蛋白质抗原的构象表位。此外，这些单克隆抗体被分为两组：对部分变性蛋白具有结合活性的和结合活性完全丧失的。接下来，当以MIHS方法作为首次筛选来筛选单克隆抗体时，我们发现通过对用荧光标记的靶抗原和荧光标记的B细胞受体抗体进行双重染色的杂交瘤，可能会筛选出具有更强结合常数的单克隆抗体。
所提出的结合MIHS和SAST的两步筛选方法，构成了一种快速、简单且有效的策略，用于获取通过杂交瘤技术产生的构象特异性单克隆抗体。本文报道的新型单克隆抗体筛选策略可加速抗体药物和抗体检测的开发。
