A Novel Anti-CDH5/VE-Cadherin Monoclonal Antibody (Ca 5 Mab-8) for Flow Cytometry,Western Blotting,and Immunohistochemistry

Abstract

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 IgG_2a, 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.