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

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.