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 crucial roles in endothelial cell adhesion, vascular barrier function, and signaling. CDH5 manages endothelial cell-cell junctions during vascular remodeling, which is vital for both vascular homeostasis and adaptive responses to pathological stimuli. Although anti-CDH5 monoclonal antibodies (mAbs) are used for specific applications such as flow cytometry, Western blotting, and immunohistochemistry (IHC), highly sensitive and versatile anti-CDH5 mAbs suitable for all these applications remain limited. Here, new anti-human CDH5 mAbs, called Ca5Mabs, were developed through a flow cytometry-based high-throughput screening. Among them, clone Ca5Mab-8 (mouse IgG2a, kappa) recognized CDH5-overexpressed Chinese hamster ovary-K1 (CHO/CDH5) cells in flow cytometry. Moreover, 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 exceeded those of a commercial anti-CDH5 mAb (clone BV9). The apparent dissociation constant of Ca5Mab-8 for CHO/CDH5 was measured as 6.1 × 10-9 M. Ca5Mab-8 can detect endogenous CDH5 in Western blotting. In addition, Ca5Mab-8, but not BV9, can be used for IHC to detect endothelial cells in formalin-fixed paraffin-embedded tissues. These findings suggest that Ca5Mab-8 is a versatile basic research tool in vascular biology and has potential for clinical applications including tumor diagnosis and therapy.