Epitope Mapping of an Anti-Mouse CXCR6 Monoclonal Antibody (Cx6 Mab-1) Using the 2 × Alanine Scanning Method

Yu Isoda, Tomohiro Tanaka, Hiroyuki Suzuki, Teizo Asano, Takuro Nakamura, Miyuki Yanaka, Saori Handa, Yu Komatsu, Saori Okuno, Nozomi Takahashi, Yuki Okada, Hiyori Kobayashi, Guanjie Li, Ren Nanamiya, Nohara Goto, Nami Tateyama, Takeo Yoshikawa, Mika K. Kaneko, Yukinari Kato

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)


The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, cytotoxic T lymphocytes (CTLs), and natural killer cells. CXCR6 plays critical roles in local expansion of effector-like CTLs in tumor microenvironment to potentiate the antitumor response. Therefore, the development of anti-CXCR6 monoclonal antibodies (mAbs) is essential to evaluate the immune microenvironment of tumors. Using N-terminal peptide immunization, we previously developed an anti-mouse CXCR6 (mCXCR6) mAb, Cx6Mab-1 (rat IgG1, kappa) , which is useful for flow cytometry and western blotting. In this study, we determined the critical epitope of Cx6Mab-1 by enzyme-linked immunosorbent assay (ELISA) using the 1 × alanine scanning (1 × Ala-scan) method or the 2 × alanine scanning (2 × Ala-scan) method. Although we first performed ELISA by 1 × Ala-scan using one alanine-substituted peptides of mCXCR6 N-terminal domain (amino acids 1-20), we could not identify the Cx6Mab-1 epitope. We next performed ELISA by 2 × Ala-scan using two alanine (or glycine) residues-substituted peptides of mCXCR6 N-terminal domain, and found that Cx6Mab-1 did not recognize S8A-A9G, A9G-L10A, L10A-Y11A, and G13A-H14A of the mCXCR6 N-terminal peptide. The results indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 of mCXCR6. Therefore, we could demonstrate that the 2 × Ala scan method is useful for determining the critical epitope of mAbs.

Original languageEnglish
Pages (from-to)275-278
Number of pages4
JournalMonoclonal antibodies in immunodiagnosis and immunotherapy
Issue number5
Publication statusPublished - 2022 Oct 1


  • 2 × Ala scanning
  • enzyme-linked immunosorbent assay
  • epitope mapping
  • monoclonal antibody
  • mouse CXCR6

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology


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