Epitope mapping of pmab-225 an anti-alpaca podoplanin monoclonal antibody using flow cytometry

Yusuke Sayama, Masato Sano, Yoshikazu Furusawa, Mika K. Kaneko, Yukinari Kato

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

Podoplanin (PDPN) is a mucin-type membrane glycoprotein, and possesses three platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 at the N-terminus of PDPN, and one or two PLAG-like domains (PLDs) in the middle of PDPN. PDPN is expressed on normal tissues, such as podocytes of the kidney and type I alveolar cells of the lung, and is also overexpressed in numerous malignant cancers. Previously, we reported a novel anti-alpaca podoplanin (aPDPN) monoclonal antibody (mAb), PMab-225, using Cell-Based Immunization and Screening (CBIS) method. PMab-225 specifically detected aPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/aPDPN) cells using flow cytometry and western blotting, and strongly stained alpaca tissues such as lung type I alveolar cells by immunohistochemistry. However, the specific binding epitope of aPDPN for PMab-225 remains unclear. Thus, in this study, a series of deletion or point mutations of aPDPN were utilized for investigating the binding epitope of PMab-225 using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-225 epitope might exist between 80 amino acid (aa) and 85 aa of aPDPN. Furthermore, the analysis of point mutants demonstrated that Thr84 of aPDPN, which exists in PLD, could be included in the critical epitope of PMab-225.

Original languageEnglish
Pages (from-to)255-260
Number of pages6
JournalMonoclonal antibodies in immunodiagnosis and immunotherapy
Volume38
Issue number6
DOIs
Publication statusPublished - 2019 Dec

Keywords

  • alpaca podoplanin
  • epitope
  • monoclonal antibody

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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