Epitope mapping of an anti-alpha thalassemia/mental retardation syndrome X-linked monoclonal antibody AMab-6

Mika K Kaneko; Shinji Yamada; Shunsuke Itai; Yoshikazu Furusawa; Takuro Nakamura; Miyuki Yanaka; Saori Handa; Kayo Hisamatsu; Yoshimi Nakamura; Masato Fukui; Hiroyuki Harada; Yukinari Kato

doi:10.1016/j.bbrep.2018.07.003

Epitope mapping of an anti-alpha thalassemia/mental retardation syndrome X-linked monoclonal antibody AMab-6

Biochem Biophys Rep. 2018 Jul 13:15:76-80. doi: 10.1016/j.bbrep.2018.07.003. eCollection 2018 Sep.

Authors

Mika K Kaneko¹, Shinji Yamada¹, Shunsuke Itai^{1

2}, Yoshikazu Furusawa^{1

3

4}, Takuro Nakamura¹, Miyuki Yanaka¹, Saori Handa¹, Kayo Hisamatsu¹, Yoshimi Nakamura¹, Masato Fukui⁴, Hiroyuki Harada², Yukinari Kato^{1

3}

Affiliations

¹ Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
² Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
³ New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
⁴ ZENOAQ RESOURCE CO., LTD., 1-1 Tairanoue, Sasagawa, Asaka-machi, Koriyama, Fukushima 963-0196, Japan.

Abstract

The alpha-thalassemia/mental-retardation-syndrome-X-linked (ATRX) gene is located on the q arm of the X chromosome. ATRX gene mutations were first discovered in pancreatic neuroendocrine tumors, and subsequently in other cancer subtypes, including gliomas. Molecular subgrouping of gliomas has been more important than conventional histological classifications. Mutations in the isocitrate dehydrogenase (IDH), telomerase reverse transcriptase (TERT) promoter, and ATRX and the codeletion of chromosomes 1p/19q are used as biomarkers for diagnosing the subtypes of diffuse gliomas. We recently developed a sensitive monoclonal antibody (mAb) AMab-6 against ATRX by immunizing mice with recombinant human ATRX. AMab-6 can help to detect ATRX mutations via Western blotting and immunohistochemical analyses. In this study, we characterized the binding epitope of AMab-6 using enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemical analysis, and found that Gln2368 of ATRX is critical for AMab-6 binding to ATRX. Our findings could be applied to the production of more functional anti-ATRX mAbs.

Keywords: AMab-6; ATRX; ATRX, alpha-thalassemia/mental-retardation-syndrome-X-linked; DAB, 3,3-diaminobenzidine tetrahydrochloride; ELISA, enzyme-linked immunosorbent assay; Epitope mapping; PBS, phosphate-buffered saline; mAb, monoclonal antibody.