Abstract
Cadherin-4 (CDH4/R-cadherin) is a type I cadherin involved in diverse processes of neural development and cancer. Although several anti-CDH4 monoclonal antibodies (mAbs) have been established for Western blotting, mAbs suitable for flow cytometric analysis of CDH4 have not been described. To generate anti-CDH4 mAbs for flow cytometry, we used a flow cytometry-based high-throughput screening strategy. Among the generated mAbs, Ca4Mab-3 (IgG1, κ) selectively recognized CDH4-overexpressed Chinese hamster ovary-K1 (CHO/CDH4) cells by flow cytometry and showed no detectable cross-reactivity with 21 other CDHs, including both type I and type II CDHs. Ca4Mab-3 also detected endogenous CDH4 in human breast cancer (MDA-MB-231) and osteosarcoma (U2OS) cell lines. The apparent dissociation constants (KD) of Ca4Mab-3 for CHO/CDH4 and U2OS cells were 1.4 × 10⁻⁹ M and 1.1 × 10⁻⁹ M, respectively. In addition, Ca4Mab-3 successfully detected endogenous CDH4 in Western blotting. These results indicate that Ca4Mab-3 is a highly specific anti-CDH4 mAb that can be used in flow cytometry and Western blotting. Ca4Mab-3 may serve as a useful tool for investigating CDH4 function and may have potential applications in the development of therapeutic strategies for CDH4-positive tumors.