Development of Sensitive Monoclonal Antibody PMab-2 Against Rat Podoplanin

Oki, Hiroharu; Honma, Ryusuke; Ogasawara, Satoshi; Fujii, Yuki; Liu, Xing; Takagi, Michiaki; Kaneko, Mika K.; Kato, Yukinari

doi:10.1089/mab.2015.0041

Cited by 61 publications

(40 citation statements)

References 42 publications

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“…(9) In this study, we produced point mutants of rPDPN using recombinant proteins and synthesized peptides and investigated the epitope of PMab-2 critical for rPDPN detection.…”

Section: Resultsmentioning

confidence: 99%

“…CHO/rPDPN was produced in our previous study. (9) The rPDPN mutation plasmids were transfected into CHO-K1 cells using Lipofectamine LTX (Thermo Fisher Scientific, Inc., Waltham, MA). CHO/rPDPN and transiently transfected cells were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37°C in a humidified atmosphere of 5% carbon dioxide and 95% air.…”

Section: Methodsmentioning

confidence: 99%

“…PMab-2 monoclonal antibody (mAb) was previously produced against PLAG domain of rPDPN (9) ; therefore, PMab-2 could neutralize interactions between rPDPN and CLEC-2. (5–7) PMab-2 is applicable in Western blot, flow cytometry, and immunohistochemical analyses.…”

Section: Introductionmentioning

confidence: 99%

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Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2

Furusawa

Yamada

Itai

et al. 2018

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Rat podoplanin (rPDPN) is a recognized lymphatic endothelial cell marker and is expressed on the podocytes of kidney and type I lung alveolar cells. rPDPN is a type I transmembrane sialoglycoprotein and induces platelet aggregation via the C-type lectin-like receptor-2 of platelets. It comprises four platelet aggregation-stimulating (PLAG) domains: PLAG1–3, present in the N-terminus, and PLAG4, in the center of the PDPN protein. Previously, we developed a mouse anti-rPDPN monoclonal antibody clone, PMab-2, by immunizing the PLAG2 and PLAG3 domains of rPDPN. PMab-2 has applications in Western blot, flow cytometry, and immunohistochemical analyses for detection of both normal and cancer cells. However, the binding epitope of PMab-2 remains to be determined. Herein, we investigated the epitope of PMab-2 using enzyme-linked immunosorbent assay, immunohistochemical analysis, and flow cytometry. The results revealed that the critical epitope of PMab-2 is Leu46 and Glu47 of rPDPN.

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“…(9) In this study, we produced point mutants of rPDPN using recombinant proteins and synthesized peptides and investigated the epitope of PMab-2 critical for rPDPN detection.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2

Furusawa

Yamada

Itai

et al. 2018

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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“…Usually, mouse MAbs are preferred for the reason that antimouse secondary antibodies are more available for almost all experiments especially for immunohistochemical analysis. For that reason, we recently produced anti-rat PDPN MAbs (41) and anti-rabbit PDPN MAbs (42,43) using mice. In this study, we tried to develop mouse anti-hPDPN MAbs, the epitope of which are similar with rat NZ-1 MAb.…”

Section: Discussionmentioning

confidence: 99%

Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin

OgasawaraSatoshi

Kaneko

HonmaRyusuke

et al. 2016

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Podoplanin (PDPN)/Aggrus is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregation-stimulating (PLAG) domain. The O-glycosylation on Thr52 of human PDPN (hPDPN) is critical for the interaction of hPDPN with C-type lectin-like receptor-2 (CLEC-2), resulting in platelet aggregation. Many anti-hPDPN monoclonal antibodies (MAbs) against PLAG domains and non-PLAG domains have been established; however, mouse anti-PLAG2/3 MAb, the epitope of which is consistent with rat anti-PLAG2/3 MAb NZ-1, has not been established. NZ-1 inhibits the hPDPN-CLEC-2 interaction and is also useful for anti-PA tag MAb. We recently established CasMab technology to produce MAbs against membranous proteins. Herein, we produced a novel anti-hPDPN MAb, LpMab-13, which binds to PLAG2/3 domains. LpMab-13 recognized endogenous hPDPN of cancer cells, including glioblastoma, oral cancer, lung cancer, and malignant mesothelioma, and normal cells such as lymphatic endothelial cells and podocytes of kidney in Western blot, flow cytometry, and immunohistochemistry. LpMab-13 recognized glycan-deficient hPDPN in flow cytometry, indicating that the interaction between LpMab-13 and hPDPN is independent of its glycosylation. The minimum epitope of LpMab-13 was identified as Ala42-Asp49 of hPDPN using Western blot and flow cytometry. The combination of different epitope-possessing MAbs could be advantageous for the hPDPN-targeting diagnosis and therapy.

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“…(17) Additionally, PMab-1 possesses high affinity and specificity toward mouse podoplanin. (18) Therefore, it was predicted that PMab-1 would have characteristics suitable for an anti-tag antibody. Here, we report the development of a novel affinity tag system comprising PMab-1 and its epitope peptide MAP tag.…”

Section: Introductionmentioning

confidence: 99%

MAP Tag: A Novel Tagging System for Protein Purification and Detection

Fujii

Kaneko

Kato

2016

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Protein purification is an essential procedure in fields such as biochemistry, molecular biology, and biophysics. Acquiring target proteins with high quality and purity is still difficult, although several tag systems have been established for protein purification. Affinity tag systems are excellent because they possess high affinity and specificity for acquiring the target proteins. Nevertheless, further affinity tag systems are needed to compensate for several disadvantages of the presently available affinity tag systems. Herein, we developed a novel affinity tag system designated as the MAP tag system. This system is composed of a rat anti-mouse podoplanin monoclonal antibody (clone PMab-1) and MAP tag (GDGMVPPGIEDK) derived from the platelet aggregation-stimulating domain of mouse podoplanin. PMab-1 possesses high affinity and specificity for the MAP tag, and the PMab-1/MAP tag complex dissociates in the presence of the epitope peptide, indicating that the MAP tag system is suitable for protein purification. We successfully purified several proteins, including a nuclear protein, soluble proteins, and a membrane protein using the MAP tag system. The MAP tag system is very useful not only for protein purification but also in protein detection systems such as western blot and flow cytometric analyses. Taken together, these findings indicate that the MAP tag system could be a powerful tool for protein purification and detection.

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Development of Sensitive Monoclonal Antibody PMab-2 Against Rat Podoplanin

Cited by 61 publications

References 42 publications

Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2

Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2

Establishment of Mouse Monoclonal Antibody LpMab-13 Against Human Podoplanin

MAP Tag: A Novel Tagging System for Protein Purification and Detection

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