TY - JOUR
T1 - Epitope mapping of pmab-225 an anti-alpaca podoplanin monoclonal antibody using flow cytometry
AU - Sayama, Yusuke
AU - Sano, Masato
AU - Furusawa, Yoshikazu
AU - Kaneko, Mika K.
AU - Kato, Yukinari
N1 - Funding Information:
This research was supported in part by AMED under grant numbers JP19am0401013 (Y.K.), JP19am0101078 (Y.K.), and JP19ae0101028 (Y.K.), and by JSPS KAKENHI grant numbers 17K07299 (M.K.K.) and 19K07705 (Y.K.).
Publisher Copyright:
© Copyright 2019, Mary Ann Liebert, Inc., publishers.
PY - 2019/12
Y1 - 2019/12
N2 - 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.
AB - 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.
KW - alpaca podoplanin
KW - epitope
KW - monoclonal antibody
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U2 - 10.1089/mab.2019.0033
DO - 10.1089/mab.2019.0033
M3 - Article
C2 - 31613697
AN - SCOPUS:85076387922
VL - 38
SP - 255
EP - 260
JO - Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
JF - Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
SN - 2167-9436
IS - 6
ER -
