Development of a specific anti-human EphA3 monoclonal antibody, Ea3Mab-20, for flow cytometry

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports Pub Date : 2025-07-04 DOI:10.1016/j.bbrep.2025.102130

Hiroyuki Satofuka, Hiroyuki Suzuki, Miu Hirose, Keisuke Shinoda, Takuya Nakamura, Tomohiro Tanaka, Mika K. Kaneko, Yukinari Kato

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引用次数: 0

Abstract

Erythropoietin-producing hepatocellular (Eph) receptor A3 (EphA3) is a member of the Eph receptor family, which binds to its respective ligands, ephrins. These interactions are essential for normal development and tissue homeostasis. Dysregulation of EphA3 has been reported to be associated with human hematopoietic malignancies, making it a promising target for therapy and diagnosis. Due to the high similarity of the extracellular domain among Eph receptors (more than 33% amino acid identity), generating highly specific monoclonal antibodies (mAbs) is crucial. We developed anti-human EphA3 mAbs in this study using the Cell-Based Immunization and Screening (CBIS) method. Among them, the clone Ea₃Mab-20 (IgG₁, kappa) exhibited high affinity and specificity in flow cytometry. The dissociation constant values of Ea₃Mab-20 for CHO/EphA3 and Jurkat cells were determined to be 9.0 ± 0.3 × 10⁻⁹ M and 1.4 ± 0.1 × 10⁻⁹ M, respectively. Ea₃Mab-20 showed no cross-reactivity with other Eph receptors in flow cytometry. Furthermore, Ea₃Mab-20 demonstrated the suitability for detecting formalin-fixed paraffin-embedded cell samples in immunohistochemistry. Therefore, Ea₃Mab-20 is valuable mAb for basic research and is expected to contribute to the clinical application of mAb for cancer therapy and diagnosis.

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用于流式细胞术的特异性抗人EphA3单克隆抗体Ea3Mab-20的研制

促红细胞生成素产生肝细胞（Eph）受体A3 （EphA3）是Eph受体家族的一员，与相应的配体ephrin结合。这些相互作用对正常发育和组织稳态至关重要。EphA3的失调已被报道与人类造血恶性肿瘤有关，使其成为治疗和诊断的一个有希望的靶点。由于Eph受体胞外结构域的高度相似性（超过33%的氨基酸同一性），产生高度特异性的单克隆抗体（mab）至关重要。本研究采用基于细胞免疫和筛选（CBIS）的方法制备了抗人EphA3单抗。其中克隆Ea3Mab-20 （IgG1, kappa）在流式细胞术中表现出较高的亲和力和特异性。测定Ea3Mab-20对CHO/EphA3和Jurkat细胞的解离常数分别为9.0±0.3 × 10−9 M和1.4±0.1 × 10−9 M。流式细胞术显示Ea3Mab-20与其他Eph受体无交叉反应性。此外，Ea3Mab-20在免疫组织化学中显示出检测福尔马林固定石蜡包埋细胞样本的适用性。因此，Ea3Mab-20是一种具有基础研究价值的mAb，有望为mAb在癌症治疗和诊断方面的临床应用做出贡献。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biochemistry and Biophysics Reports Biochemistry, Genetics and Molecular Biology-Biophysics

CiteScore

4.60

自引率

0.00%

发文量

191

审稿时长

59 days

期刊介绍： Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.