Tanaka, T.; Tawara, M.; Suzuki, H.; Kaneko, M.K.; Kato, Y. Identification of the Binding Epitope of an Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) Using 1× Alanine Scanning. Antibodies2023, 12, 32.
Tanaka, T.; Tawara, M.; Suzuki, H.; Kaneko, M.K.; Kato, Y. Identification of the Binding Epitope of an Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) Using 1× Alanine Scanning. Antibodies 2023, 12, 32.
Tanaka, T.; Tawara, M.; Suzuki, H.; Kaneko, M.K.; Kato, Y. Identification of the Binding Epitope of an Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) Using 1× Alanine Scanning. Antibodies2023, 12, 32.
Tanaka, T.; Tawara, M.; Suzuki, H.; Kaneko, M.K.; Kato, Y. Identification of the Binding Epitope of an Anti-Mouse CCR6 Monoclonal Antibody (C6Mab-13) Using 1× Alanine Scanning. Antibodies 2023, 12, 32.
Abstract
CC chemokine receptor 6 (CCR6) is one of the members of G protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, including B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. Coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, autoimmune diseases, and psoriasis. Therefore, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for patients. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6Mab-13 (rat IgG1, kappa), applicable for flow cytometry by immunizing a rat with N-terminal peptide of mCCR6. This study investigated the binding epitope of C6Mab-13 using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) methods with the synthesized point mutated-peptides within 1-20 amino acids region of mCCR6. In ELISA, C6Mab-13 lost the reaction to the alanine-substituted peptide of D11A. The epitope of C6Mab-13 was identified to be Asp11 in ELISA. Furthermore, in SPR analysis, the dissociation constants (KD) could not be calculated for G9A and D11A mutants due to lack of binding. The SPR analysis demonstrated that the C6Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C6Mab-13 was determined to be around Asp11 on mCCR6. Based on the epitope information, C6Mab-13 could be useful for further functional analysis of mCCR6 in future studies.
Biology and Life Sciences, Biochemistry and Molecular Biology
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