Ea8Mab-9: A novel monoclonal antibody against erythropoietin-producing hepatocellular receptor A8 for flow cytometry

Erythropoietin-producing hepatocellular receptor A8 (EphA8) is a type I transmembrane protein that belongs to the largest erythropoietin-producing hepatocellular (Eph) family among receptor tyrosine kinases. By binding to its membrane-bound ephrin-A or ephrin-B ligands on adjacent cells, Eph receptors form complexes and mediate bidirectional signaling activities, triggering cell-cell adhesion and repulsion. Increased expression of EphA8 correlates with poor prognosis in some types of cancer. Therefore, developing sensitive monoclonal antibodies (mAbs) for EphA8 has been desired for treatment, diagnosis, and further basic research. In particular, there are no anti-EphA8 mAbs that can be used for flow cytometry. A novel, specific, and sensitive anti-human EphA8 mAb, which applies to flow cytometry, clone Ea8Mab-9 (mouse immunoglobulin G1, kappa), was established using the Cell-Based Immunization and Screening method. Ea8Mab-9 reacted with EphA8-overexpressed Chinese hamster ovary-K1 cells (CHO/EphA8) and EphA8-overexpressed LN229 glioblastoma cells (LN229/EphA8) in flow cytometry. Notably, Ea8Mab-9 did not recognize other members of the Eph receptor family. Furthermore, Ea8Mab-9 demonstrated a high binding affinity for CHO/EphA8 and LN229/EphA8, with dissociation constants of 1.3 × 10-9 M and 1.6 × 10-9 M, respectively. The reaction of Ea8Mab-9 with CHO/EphA8 was completely blocked by a recombinant EphA8 protein. Ea8Mab-9 could be useful for analyzing the EphA8-related biological responses using flow cytometry, owing to its high affinity and specificity.