A Novel Anti-CDH5/VE‐Cadherin Monoclonal Antibody (Ca5Mab-8) for Flow Cytometry, Western Blotting, and Immunohistochemistry

Cadherin-5 (CDH5), also known as vascular endothelial cadherin (VE-cadherin), plays essential roles in endothelial cell adhesion, vascular barrier function, and signaling. CDH5 coordinates endothelial cell–cell junction during vascular remodeling, which is indispensable for both vascular homeostasis and adaptive responses to pathological stimuli. Although anti-CDH5 monoclonal antibodies (mAbs) can be used for individual applications including flow cytometry, western blotting, and immunohistochemistry (IHC), highly sensitive and versatile anti-CDH5 mAbs for all applications remain limited. Here, novel anti-human CDH5 mAbs, designated Ca₅Mabs, were developed using a flow cytometry-based high-throughput screening. Among them, a clone Ca₅Mab-8 (IgG_2a, κ) recognized CDH5-overexpressed Chinese hamster ovary-K1 (CHO/CDH5) cells in flow cytometry. Furthermore, Ca₅Mab-8 also recognized endogenous CDH5-expressing human endothelial cell lines (HUVEC/TERT2 and HDMVEC/TERT164-B) and a cervical cancer cell line (Hela). These reactivities were superior to a commercially available anti-CDH5 mAb (clone BV9). The dissociation constant value of Ca₅Mab-8 for CHO/CDH5 was determined as 6.1 × 10⁻⁹ M. Ca₅Mab-8 can detect endogenous CDH5 in Western blotting. Moreover, Ca₅Mab-8, but not BV9, is available for IHC to detect endothelial cells in formalin-fixed paraffin-embedded tissues. These results indicate that Ca₅Mab-8 is versatile for research and are expected to contribute to clinical applications, such as tumor diagnosis and therapy.