TY - JOUR
T1 - Development and Characterization of Anti-Sheep Podoplanin Monoclonal Antibodies PMab-253 and PMab-260
AU - Kaneko, Mika K.
AU - Sano, Masato
AU - Takei, Junko
AU - Asano, Teizo
AU - Sayama, Yusuke
AU - Hosono, Hideki
AU - Kobayashi, Atsushi
AU - Konnai, Satoru
AU - Kato, Yukinari
N1 - Funding Information:
This research was supported, in part, by Japan Agency for Medical Research and Development (AMED) under Grant Numbers: JP20am0401013 (Y.K.), JP20am0101078 (Y.K.), and JP20ae0101028 (Y.K.), and by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers: 17K07299 (M.K.K.), 19K07705 (Y.K.), and 20K16322 (M.S.).
Publisher Copyright:
© Copyright 2020, Mary Ann Liebert, Inc., publishers 2020.
PY - 2020/8/1
Y1 - 2020/8/1
N2 - Anti-podoplanin (PDPN) monoclonal antibodies (mAbs) are needed as markers for lymphatic endothelial cells or type I alveolar cells in immunohistochemical analyses. We have developed anti-PDPN mAbs for many species, including humans, mice, rats, rabbits, dogs, cats, bovines, pigs, Tasmanian devils, alpacas, tigers, whales, goats, horses, and bears. This study develops and characterizes anti-sheep PDPN (sPDPN) mAbs using Cell-Based Immunization and Screening (CBIS) method. A RAP14 tag was added to the N-terminus of sPDPN, and an anti-RAP14 tag mAb (PMab-2) was used to measure the expression level of sPDPN in flow cytometry and Western blots. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. Two of the mAbs, PMab-253 (immunoglobulin M [IgM], kappa) and PMab-260 (IgM, kappa), detected CHO/sPDPN cells specifically using flow cytometry and Western blots. Both PMab-253 and PMab-260 stained the renal glomerulus and Bowman's capsule, lymphatic endothelial cells of the lung and colon, and type I alveolar cells of the lung, suggesting PMab-253 and PMab-260, which were developed by CBIS method, can be applied to functional analyses of sPDPN. We also determined the binding epitope of PMab-253 and PMab-260 using flow cytometry. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-253 and PMab-260 epitope exists between amino acids 110 and 115 of sPDPN. Analysis of sPDPN point mutations revealed that the critical epitope of PMab-253 and PMab-260 includes Thr112 and Ser113 of sPDPN, indicating that the PMab-253 and PMab-260 epitope are independent of the platelet aggregation-stimulating (PLAG) domain or the PLAG-like domain of sPDPN.
AB - Anti-podoplanin (PDPN) monoclonal antibodies (mAbs) are needed as markers for lymphatic endothelial cells or type I alveolar cells in immunohistochemical analyses. We have developed anti-PDPN mAbs for many species, including humans, mice, rats, rabbits, dogs, cats, bovines, pigs, Tasmanian devils, alpacas, tigers, whales, goats, horses, and bears. This study develops and characterizes anti-sheep PDPN (sPDPN) mAbs using Cell-Based Immunization and Screening (CBIS) method. A RAP14 tag was added to the N-terminus of sPDPN, and an anti-RAP14 tag mAb (PMab-2) was used to measure the expression level of sPDPN in flow cytometry and Western blots. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. Two of the mAbs, PMab-253 (immunoglobulin M [IgM], kappa) and PMab-260 (IgM, kappa), detected CHO/sPDPN cells specifically using flow cytometry and Western blots. Both PMab-253 and PMab-260 stained the renal glomerulus and Bowman's capsule, lymphatic endothelial cells of the lung and colon, and type I alveolar cells of the lung, suggesting PMab-253 and PMab-260, which were developed by CBIS method, can be applied to functional analyses of sPDPN. We also determined the binding epitope of PMab-253 and PMab-260 using flow cytometry. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-253 and PMab-260 epitope exists between amino acids 110 and 115 of sPDPN. Analysis of sPDPN point mutations revealed that the critical epitope of PMab-253 and PMab-260 includes Thr112 and Ser113 of sPDPN, indicating that the PMab-253 and PMab-260 epitope are independent of the platelet aggregation-stimulating (PLAG) domain or the PLAG-like domain of sPDPN.
KW - monoclonal antibody
KW - podoplanin
KW - sPDPN
KW - sheep
UR - http://www.scopus.com/inward/record.url?scp=85089819538&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85089819538&partnerID=8YFLogxK
U2 - 10.1089/mab.2020.0018
DO - 10.1089/mab.2020.0018
M3 - Article
C2 - 32679010
AN - SCOPUS:85089819538
VL - 39
SP - 144
EP - 155
JO - Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
JF - Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
SN - 2167-9436
IS - 4
ER -