Establishment of a Sensitive Monoclonal Antibody Against Mouse CCR9 (C₉Mab-24) for Flow Cytometry

The CC chemokine receptor 9 (CCR9), also known as CD199, is one of chemokine receptors. The CC chemokine ligand 25 (CCL25) is known to be the only ligand for CCR9. The CCR9-CCL25 interaction plays important roles in chemotaxis of lymphocytes and tumor cell migration. Therefore, CCR9-CCL25 axis is a promising target for tumor therapy and diagnosis. In this study, we established a sensitive and specific monoclonal antibody (mAb) against mouse CCR9 (mCCR9) using N-terminal peptide immunization method. The established anti-mCCR9 mAb, C₉Mab-24 (rat immunoglobulin [IgG]_2a, kappa), reacted with mCCR9-overexpressed Chinese hamster ovary-K1 (CHO/mCCR9) and mCCR9-endogenously expressed cell line, RL2, through flow cytometry. Kinetic analyses using flow cytometry showed that the dissociation constants (K_D) of C₉Mab-24 for CHO/mCCR9 and RL2 cell lines were 6.0 × 10^-9 M and 4.7 × 10^-10 M, respectively. Results indicated that C₉Mab-24 is useful for detecting mCCR9 through flow cytometry, thereby providing a possibility for targeting mCCR9-expressing cells in vivo experiments.