Development of a Novel Anti-human EphA1 Monoclonal Antibody, EaMab-30, for Multiple Applications.

Erythropoietin-producing hepatocellular receptor A1 (EphA1) is one of the Eph receptor family members, the largest group of receptor tyrosine kinases. EphA1 is expressed in various tissues and regulates cellular homeostasis by interacting with its membrane-bound ephrin ligands and other receptors. EphA1 critically correlates with the pathogenesis in several disorders, including Alzheimer's disease and cancers. Therefore, establishing sensitive monoclonal antibodies (mAbs) for EphA1 has been desired for basic research, diagnosis, and treatment. In this study, a novel specific and sensitive anti-human EphA1 mAb, clone EaMab-30 (mouse IgG, kappa), was established by the Cell-Based Immunization and Screening (CBIS) method. EaMab-30 demonstrated reactivity with an EphA1-overexpressed Chinese hamster ovary-K1 cell line (CHO/EphA1), an endogenously EphA1-expressing bladder carcinoma cell line (5637), and a colorectal adenocarcinoma cell line (Caco-2) in flow cytometry. Crossreactivities of EaMab-30 with other Eph receptors were not observed. Furthermore, the values of apparent binding affinity for CHO/EphA1 and 5637 were determined to be 8.9 × 10 M and 1.7 × 10 M, respectively. Furthermore, EaMab-30 detected EphA1 protein in CHO/EphA1 and 5637 lysates using Western blot analysis. EaMab-30 also clearly stained EphA1 of formalin-fixed paraffin-embedded CHO/EphA1 using immunohistochemistry. EaMab-30, established by CBIS method, could help analyze the EphA1-contributed cellular functions and have potential applications in pathological diagnosis and treatment with specificity and high affinity for EphA1-expressing cells.