Ea5Mab-7, a Novel Anti-EphA5 Monoclonal Antibody for Flow Cytometry and Immunohistochemistry

Eph receptor A5 (EphA5) binds to ephrin ligands and initiate the intracellular signaling through its tyrosine kinase activity. EphA5 is upregulated or mutated in various tumors, which is associated with the tumor progression and the sensitivity to immune therapy, respectively. Therefore, EphA5 has been proposed as an attractive target for tumor diagnosis and therapy. Thus, monoclonal antibody (mAb) against EphA5 is essential for basic research and development for mAb-based tumor therapy. In this study, we developed a novel anti-human EphA5 mAb, Ea5Mab-7, using the Cell-Based Immunization and Screening (CBIS) method. Ea5Mab-7 reacted with EphA5-overexpressed Chinese hamster ovary-K1 (CHO/EphA5), EphA5-overexpressed glioblastoma LN229 (LN229/EphA5), and endogenous EphA5-positive osteosarcoma Saos-2 in flow cytometry. The binding affinities (KD values) were determined as 3.2 × 10-9 M for CHO/EphA5, 3.0 × 10-9 M for LN229/EphA5, and 1.3 × 10-8M for Saos-2. Furthermore, Ea5Mab-7 did not exhibit cross-reactivity with other Eph receptor-overexpressed CHO-K1. In addition, Ea5Mab-7 is suitable for immunohistochemistry. These results indicate that Ea5Mab-7, established using the CBIS method, facilitates basic studies of EphA5 and is expected to be useful for mAb-based tumor diagnosis and therapy.