MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 5, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0022

Characterization of Anti-Goat Podoplanin Monoclonal
Antibody PMab-235 Using Immunohistochemistry
Against Goat Tissues

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Junko Takei,1,2 Shunsuke Itai,1,2 Hiroyuki Harada,2 Yoshikazu Furusawa,1,3,4 Takashi Miwa,4 Masato Fukui,4Takuro Nakamura,1 Masato Sano,1 Yusuke Sayama,1 Miyuki Yanaka,1 Saori Handa,1 Kayo Hisamatsu,1
Yoshimi Nakamura,1 Shinji Yamada,1 Mika K. Kaneko,1 and Yukinari Kato1,3

Podoplanin (PDPN)/T1alpha is expressed on lymphatic endothelial cells, type I alveolar cells of the lungs, andpodocytes of the kidney. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1,
PLAG2, and PLAG3) of the N-terminus and the PLAG-like domains (PLDs). We previously reported an antigoat PDPN (gPDPN) monoclonal antibody (mAb), PMab-235, which was developed using the Cell-Based
Immunization and Screening (CBIS) method. PMab-235 is very useful in flow cytometry, Western blotting, andimmunohistochemical analyses; however, the binding epitope of PMab-235 remains to be elucidated. In thisstudy, we investigated the epitopes of PMab-235 using enzyme-linked immunosorbent assay and immunohistochemistry. The results revealed that the critical epitope of PMab-235 produced by CBIS method is Arg75,
Leu78, and Pro79 of gPDPN, which is included in PLD. The findings of our study can be applied to the
production of more functional anti-gPDPN mAbs.
Keywords: podoplanin, PDPN, PMab-235, epitope mapping

IntroductionP

odoplanin (PDPN)/T1alpha is a type I transmembrane glycoprotein, which is expressed in normal tissues, such as type I lung alveolar cells, renal corpuscles, andlymphatic endothelial cells.(1,2) PDPN induces platelet aggregation through C-type lectin-like receptor-2 (CLEC-2) on
platelets.(1,3–9) The PDPN/CLEC-2 interaction facilitates theseparation of embryonic blood/lymphatic vessels.(10) The expression of human PDPN was reported in several malignant
tumors, such as malignant brain tumors,(11–14) oral squamouscell carcinomas,(15) malignant mesotheliomas,(16,17) andlung cancers.(18) PDPN expression has also been associatedwith malignant progression and cancer metastasis.(6,11,19)
Because pathophysiological studies of alveolar cells of thelungs, podocytes of the kidneys, or lymphatic endothelial
cells of the colon using anti-goat PDPN (gPDPN) mAbs areimportant in many disorders, we used the Cell-Based Immunization and Screening (CBIS) method and developed
specific and sensitive monoclonal antibodies (mAbs) against
the gPDPN of 168 amino acids to facilitate the immunohis-

tochemical analysis of paraffin-embedded tissue sections.(20)PMab-235 recognized CHO/gPDPN cells, but showed noreaction with CHO-K1 cells, as assessed by flow cytometry.
PMab-235 cross-reacts with bovine PDPN. In contrast, PMab235 did not react with human, mouse, rat, rabbit, dog, cat, pig,
horse, Tasmanian devil, sheep, alpaca, tiger, whale, or bear
PDPNs. KD of PMab-235 for CHO/gPDPN cells was determined to be 1.5 · 10-8, indicating a moderate affinity ofPMab-235 for CHO/gPDPN cells. The immunohistochemical
analyses revealed that PMab-235 strongly stained type I alveolar cells of lung, podocytes of kidney, and lymphatic endothelial cells of colon. This study aimed to determine thebinding epitope of PMab-235 to gPDPN using enzyme-linked
immunosorbent assay (ELISA) and immunohistochemistry.
Materials and Methods
Enzyme-linked immunosorbent assay

Synthesized gPDPN peptides using PEPScreen (SigmaAldrich Corp., St. Louis, MO) were immobilized on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientific,

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
3
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
4
ZENOAQ RESOURCE CO., LTD., Koriyama, Japan.
2

213

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TAKEI ET AL.

Table 1. Determination of PMab-235 Epitopeby Enzyme-Linked Immunosorbent Assay
PeptideSequence

PMab-235

pp21-40
pp31-50
pp41-60
pp51-70
pp61-80
pp71-90
pp81-100
pp91-110
pp101-120
pp111-130
pp121-135

EGASTVRPEDDVTTGVKNEQ
DVTTGVKNEQTTLGVEDYMTTTLGVEDYMTTPAASKESLATPAASKESLATPMPTGTENVTPMPTGTENVTHGHREDLPTTHGHREDLPTAESTTAKSTPAESTTAKSTPAESTPAKSTPAESTPAKSTPAKSTVHTHVPAKSTVHTHVPATSHSQGKTDATSHSQGKTDGEKPKSTQKGGEKPKSTQKGGFSVG+++-

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+++, OD655S0.6; -, OD655<0.1

Inc., Waltham, MA) at 10 mg/mL for 30 minutes at 37C.
After blocking with SuperBlock T20 (PBS) Blocking Buffer(Thermo Fisher Scientific, Inc.), the plates were incubatedwith purified PMab-235 (1 mg/mL), followed by a peroxidaseconjugated anti-mouse IgG (Agilent Technologies, Inc., Santa
Clara, CA) dilution of 1:2000. Enzymatic reactions were
performed using 1-Step Ultra TMB-ELISA (Thermo Fisher
Scientific, Inc.). Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc.,
Berkeley, CA). These reactions were performed at 37C using
a total sample volume of 50–100 mL.

Immunohistochemical analyses

Goat tissues (lungs and colons) were previously collected
in NIPPON ZENYAKU KOGYO CO., LTD. (Fukushima,
Japan), and fixed in 4% paraformaldehyde phosphate buffer
solution (Nacalai Tesque, Inc., Kyoto, Japan).(21) The tissues
were processed to produce 4-mm paraffin-embedded tissue
sections, which were directly autoclaved in citrate buffer(pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20
minutes. These tissue sections were then blocked with theSuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher
Scientific, Inc.), incubated with PMab-235 (0.01–1 mg/mL)
for 1 hour at room temperature, and treated using an Envision +Kit (Agilent Technologies, Inc.) for 30 minutes. Color wasdeveloped with 3,3¢-diaminobenzidine tetrahydrochloride(DAB; Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed using hematoxylin (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan).
Blocking assay using immunohistochemical analyses

Goat tissue sections were blocked with the SuperBlock
T20 (PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.),
incubated with PMab-235 (0.2 or 0.02 mg/mL) or PMab-235(0.2 or 0.02 mg/mL) + peptides (1 mg/mL) for 1 hour at room
temperature, and treated using an Envision + Kit (Agilent
Technologies, Inc.) for 30 minutes. Color was developedwith DAB (Agilent Technologies, Inc.) for 2 minutes, andcounterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

FIG. 1. Epitope mapping using synthetic peptides of gPDPN. Synthesized gPDPN peptides were immobilized at
10 mg/mL, and were incubated with PMab-235, followed by secondary antibodies. Black bar: positive reaction by PMab235. gPDPN, goat podoplanin.

EPITOPE OF PMAB-235
Resultslung (Figs. 3E, F), indicating that 61–80 peptide is an
epitope of PMab-235.
PMab-235 also showed very high reaction with lymphatic
endothelial cells of goat colons in a dose-dependent manner (0.1–1 mg/mL; Fig. 4), even at a low concentration of
0.1 mg/mL (Fig. 4G, H). We performed a blocking assay
using immunohistochemistry against goat colons at a concentration of 0.2 mg/mL. PMab-235 reacted with lymphatic

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We first synthesized a series of gPDPN peptides, which aresummarized in Table 1. Using ELISA, PMab-235 detected a
61–80 peptide (61-TPMPTGTENVTHGHREDLPT-80) of
gPDPN, whereas it did not react with the other peptides: 21–
40, 31–50, 41–60, 51–70, 71–90, 81–100, 91–110, 101–120,
111–130, and 121–135 peptides (Fig. 1 and Table 1).
We next moved on to the blocking assay to confirm thebinding epitope of PMab-235. We first performed immunohistochemical analyses for goat lung tissues. PMab-235
showed very high reaction with type I alveolar cells ofgoat lungs in a dose-dependent manner (0.01–0.1 mg/mL;
Fig. 2). Then, we performed a blocking assay using immunohistochemistry against goat lungs at a concentrationof 0.02 mg/mL. PMab-235 reacted with type I alveolar cellsof lung (Figs. 3A, B). These reactions were not neutralized
by 21–40 peptide in lung (Figs. 3C, D). In contrast, thesereactions were partially neutralized by 61–80 peptide in

215

FIG. 2. Immunohistochemical analyses against goatlung tissues using PMab-235. Histological sections of thegoat lungs were directly autoclaved in citrate buffer for
20 minutes. After blocking with SuperBlock T20 (PBS)
Blocking Buffer, the sections were incubated with PMab235 at a concentration of 0.1 mg/mL (A, B), 0.05 mg/mL(C, D), 0.02 mg/mL (E, F), and 0.01 mg/mL (G, H). Bar:
100 mm.

FIG. 3. Immunohistochemical analyses against goat lungtissues using PMab-235 and peptides of gPDPN. Histologicalsections of the goat lungs were directly autoclaved in citratebuffer for 20 minutes. After blocking with SuperBlock T20(PBS) Blocking Buffer, the sections were incubated with (A, B)
PMab-235 (0.02 mg/mL), (C, D) PMab-235 (0.02 mg/mL) +21–40 (1 mg/mL), (E, F) PMab-235 (0.02 mg/mL) + 61–80(1 mg/mL), or (G, H) control (blocking buffer), followed bytreatment with the Envision + Kit. (I, J). Bar: 100 mm. HE,
hematoxylin and eosin.

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216

TAKEI ET AL.

FIG. 4. Immunohistochemical analyses against goat colon
tissues using PMab-235. Histological sections of the goatcolons were directly autoclaved in citrate buffer for 20
minutes. After blocking with SuperBlock T20 (PBS)
Blocking Buffer, the sections were incubated with PMab235 at a concentration of 1 mg/mL (A, B), 0.5 mg/mL (C, D),
0.2 mg/mL (E, F), and 0.1 mg/mL (G, H). Bar: 100 mm.

endothelial cells of colons (Figs. 5A, B). These reactions
were not neutralized by 21–40 peptide (Figs. 5C, D). In
contrast, these reactions were completely neutralized by 61–
80 peptide (Figs. 5E, F), also confirming that 61–80 peptide
is an epitope of PMab-235.
We then synthesized point mutants of 61–80 peptides(Table 2). Using ELISA, PMab-235 detected T61A, P62A,
M63A, P64A, T65A, G66A, T67A, E68A, N69A, V70A,
T71A, H72A, G73A, H74A, E76A, D77A, and T80A. Conversely, it did not detect R75A, L78A, and P79A, indicatingthat R75A, L78A, and P79A are critical for PMab-235 todetect gPDPN.
Discussion

We previously developed mAbs against human,(22)mouse,(22) rat,(23) rabbit,(24) dog,(25) cat,(26) bovine,(27)horse,(28–30) and pig PDPNs.(31,32) PDPN comprises threetandem-repeat of the ‘‘EDxxVTPG’’ sequences, which were
defined to be platelet aggregation-stimulating (PLAG) do-

FIG. 5. Immunohistochemical analyses against goatcolon tissues using PMab-235 and peptides of gPDPN.
Histological sections of the goat colons were directly autoclaved in citrate buffer for 20 minutes. After blocking
with SuperBlock T20 (PBS) Blocking Buffer, the sections
were incubated with (A, B) PMab-235 (0.2 mg/mL), (C, D)
PMab-235 (0.2 mg/mL) + 21–40 (1 mg/mL), (E, F) PMab235 (0.2 mg/mL) + 61–80 (1 mg/mL), or (G, H) control(blocking buffer), followed by treatment with the Envision + Kit. (I, J). Red arrows indicate lymphatic endothelial cells. Bar: 100 mm.
mains (PLAG1, PLAG2, and PLAG3) of the N-terminus of the
PDPN protein.(4) There are also several PLAG-like domains(PLDs) of the ‘‘E(D/E)xx(T/S)xx’’ sequence. PLDs, one ofwhich is also named as PLAG4, are reportedly important for
PDPN–CLEC-2 interactions.(33) Almost all mAbs against
PDPNs reportedly react with PLAG domains or PLDs.(33,34)
As summarized in Figure 6, PMab-235 detected PLD ofgPDPN.

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EPITOPE OF PMAB-235

217

FIG. 6. Summary of PMab-235 epitope. (A–C) Immunohistochemical analyses against goat lung tissues using PMab-235
and peptides of gPDPN. Each picture is the enlargement of type I alveolar cells of Figure 3. (D–F) Immunohistochemical
analyses against lymphatic endothelial cells of colon using PMab-235 and peptides of gPDPN. Each picture is the enlargement of lymphatic endothelial cells of Figure 5. Bar: 100 mm. (G) Schematic illustration of the epitope recognized by
PMab-235. PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain.
Table 2. Determination of PMab-235 Epitopeby Enzyme-Linked Immunosorbent Assay
Mutation

Sequence

PMab-235

T61A
P62A
M63A
P64A
T65A
G66A
T67A
E68A
N69A
V70A
T71A
H72A
G73A
H74A
R75A
E76A
D77A
L78A
P79A
T80A

APMPTGTENVTHGHREDLPTTAMPTGTENVTHGHREDLPTTPAPTGTENVTHGHREDLPTTPMATGTENVTHGHREDLPTTPMPAGTENVTHGHREDLPTTPMPTATENVTHGHREDLPTTPMPTGAENVTHGHREDLPTTPMPTGTANVTHGHREDLPTTPMPTGTEAVTHGHREDLPTTPMPTGTENATHGHREDLPTTPMPTGTENVAHGHREDLPTTPMPTGTENVTAGHREDLPTTPMPTGTENVTHAHREDLPTTPMPTGTENVTHGAREDLPTTPMPTGTENVTHGHAEDLPTTPMPTGTENVTHGHRADLPTTPMPTGTENVTHGHREALPTTPMPTGTENVTHGHREDAPTTPMPTGTENVTHGHREDLATTPMPTGTENVTHGHREDLPA

+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++

+++, OD655S0.6; -, OD655<0.1

PMab-235
cross-reactedwithbovinePDPN(bovPDPN).(20) The corresponding sequence of PMab235 epitope ‘‘REDLPT’’ is also ‘‘REDLPT’’ about
bovPDPN, which indicates 100% identity between gPDPN
and bovPDPN. In the blocking assay using immunohistochemistry against goat lungs, the 61–80 peptide of goat
PDPN did not completely block the PMab-235 binding totype I alveolar cells (Figs. 3E, F) because PMab-235 wasdeveloped using CBIS method, not by immunizing synthetic peptides, then the binding affinity of PMab-235
against type I alveolar cells might be higher than that forthe 61–80 peptide.
Taken together, PLD of gPDPN was clarified to be advantageous epitopes for several applications, such as flow
cytometry, Western blotting, and immunohistochemical analyses. These findings can be applied to the production of
more functional anti-gPDPN mAbs.
Acknowledgments

This research was supported in part by AMED underGrant Nos. JP18am0101078 (Y.K.), JP18am0301010(Y.K.), and JP18ae0101028 (Y.K.), and by JSPS KAKENHI
Grant Nos. 17K07299 (M.K.K.) and 19K07705 (Y.K.).

218
Author Disclosure Statement

Y.K. received research funding from ZENOAQ RESOURCE CO., LTD. The other authors have no conflict ofinterest.

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References

1. Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P,
Poczewski H, Kalt R, Schaffner G, and Kerjaschki D: Podoplanin, novel 43-kd membrane protein of glomerularepithelial cells, is down-regulated in puromycin nephrosis.
Am J Pathol 1997;151:1141–1152.
2. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R,
Amann G, Kriehuber E, Diem K, Weninger W, Tschachler
E, Alitalo K, and Kerjaschki D: Angiosarcomas express
mixed endothelial phenotypes of blood and lymphatic
capillaries: Podoplanin as a specific marker for lymphatic
endothelium. Am J Pathol 1999;154:385–394.
3. Kato Y, Fujita N, Kunita A, Sato S, Kaneko M, Osawa M,
and Tsuruo T: Molecular identification of Aggrus/T1alphaas a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem 2003;278:51599–51605.
4. Kaneko MK, Kato Y, Kitano T, and Osawa M: Conservationof a platelet activating domain of Aggrus/podoplanin as aplatelet aggregation-inducing factor. Gene 2006;378:52–57.
5. Martin-Villar E, Scholl FG, Gamallo C, Yurrita MM,
Munoz-Guerra M, Cruces J, and Quintanilla M: Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamouscell carcinomas. Int J Cancer 2005;113:899–910.
6. Kato Y, Kaneko MK, Kunita A, Ito H, Kameyama A,
Ogasawara S, Matsuura N, Hasegawa Y, Suzuki-Inoue K,
Inoue O, Ozaki Y, and Narimatsu H: Molecular analysis of
the pathophysiological binding of the platelet aggregationinducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 2008;99:54–61.
7. Kaneko MK, Kunita A, Abe S, Tsujimoto Y, Fukayama M,
Goto K, Sawa Y, Nishioka Y, and Kato Y: Chimeric antipodoplanin antibody suppresses tumor metastasis throughneutralization and antibody-dependent cellular cytotoxicity.
Cancer Sci 2012;103:1913–1919.
8. Suzuki-Inoue K, Kato Y, Inoue O, Kaneko MK, Mishima
K, Yatomi Y, Yamazaki Y, Narimatsu H, and Ozaki Y:
Involvement of the snake toxin receptor CLEC-2, in
podoplanin-mediated platelet activation, by cancer cells.
J Biol Chem 2007;282:25993–26001.
9. Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK,
Kato Y, and Yamaguchi Y: A Platform of C-Type lectinlike receptor CLEC-2 for binding O-glycosylated podoplanin and nonglycosylated rhodocytin. Structure 2014;22:
1711–1721.
10. Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z,
Chen M, Chen CY, Xu B, Lu MM, Zhou D, Sebzda E,
Santore MT, Merianos DJ, Stadtfeld M, Flake AW, Graf T,
Skoda R, Maltzman JS, Koretzky GA, and Kahn ML:
Platelets regulate lymphatic vascular development through
CLEC-2-SLP-76 signaling. Blood 2010;116:661–670.
11. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T, andMatsutani M: Increased expression of podoplanin in malignant
astrocytic tumors as a novel molecular marker of malignant
progression. Acta Neuropathol (Berl) 2006;111:483–488.
12. Mishima K, Kato Y, Kaneko MK, Nakazawa Y, Kunita A,
Fujita N, Tsuruo T, Nishikawa R, Hirose T, and Matsutani

TAKEI ET AL.

M: Podoplanin expression in primary central nervous system germ cell tumors: A useful histological marker for the
diagnosis of germinoma. Acta Neuropathol (Berl) 2006;
111:563–568.
13. Kato Y, Vaidyanathan G, Kaneko MK, Mishima K, Srivastava N, Chandramohan V, Pegram C, Keir ST, Kuan
CT, Bigner DD, and Zalutsky MR: Evaluation of antipodoplanin rat monoclonal antibody NZ-1 for targeting
malignant gliomas. Nucl Med Biol 2010;37:785–794.
14. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
15. Ochoa-Alvarez JA, Krishnan H, Pastorino JG, Nevel E,
Kephart D, Lee JJ, Retzbach EP, Shen Y, Fatahzadeh M,
Baredes S, Kalyoussef E, Honma M, Adelson ME, Kaneko
MK, Kato Y, Young MA, Deluca-Rapone L, Shienbaum
AJ, Yin K, Jensen LD, and Goldberg GS: Antibody andlectin target podoplanin to inhibit oral squamous carcinoma
cell migration and viability by distinct mechanisms. Oncotarget 2015;6:9045–9060.
16. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
17. Abe S, Morita Y, Kaneko MK, Hanibuchi M, Tsujimoto Y,
Goto H, Kakiuchi S, Aono Y, Huang J, Sato S, Kishuku M,
Taniguchi Y, Azuma M, Kawazoe K, Sekido Y, Yano S,
Akiyama S, Sone S, Minakuchi K, Kato Y, and Nishioka Y:
A novel targeting therapy of malignant mesothelioma using
anti-podoplanin antibody. J Immunol 2013;190:6239–6249.
18. Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, and Osawa
M: Enhanced expression of Aggrus (T1alpha/podoplanin),a platelet-aggregation-inducing factor in lung squamouscell carcinoma. Tumor Biol 2005;26:195–200.
19. Kunita A, Kashima TG, Morishita Y, Fukayama M, Kato
Y, Tsuruo T, and Fujita N: The platelet aggregationinducing factor aggrus/podoplanin promotes pulmonary
metastasis. Am J Pathol 2007;170:1337–1347.
20. Furusawa Y, Yamada S, Nakamura T, Sano M, Itai S, Takei
J, Harada H, Fukui M, Kaneko MK, and Kato Y: PMab235: A monoclonal antibody for immunohistochemical
analysis against goat podoplanin. Heliyon 2019;5:e02063.
21. Yamada S, Kaneko MK, Furusawa Y, Itai S, Sano M,
Nakamura T, Yanaka M, Handa S, Hisamatsu K, Nakamura
Y, Koyanagi M, Fukui M, Harada H, and Kato Y: Antibovine podoplanin monoclonal antibody PMab-44 detects
goat podoplanin in immunohistochemistry. Monoclon Antib Immunodiagn Immunother 2019;38:96–99.
22. Kaji C, Tsujimoto Y, Kato Kaneko M, Kato Y, and Sawa
Y: Immunohistochemical examination of novel rat monoclonal antibodies against mouse and human podoplanin.
Acta Histochem Cytochem 2012;45:227–237.
23. Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Takagi M,
Kaneko MK, and Kato Y: Development of sensitive
monoclonal antibody PMab-2 against rat podoplanin.
Monoclon Antib Immunodiagn Immunother 2015;34:
396–403.
24. Honma R, Fujii Y, Ogasawara S, Oki H, Liu X, Nakamura
T, Kaneko MK, Takagi M, and Kato Y: Establishment of anovel monoclonal antibody PMab-32 against rabbit podoplanin. Monoclon Antib Immunodiagn Immunother 2016;35:41–47.
25. Honma R, Kaneko MK, Ogasawara S, Fujii Y, Konnai S,
Takagi M, and Kato Y: Specific detection of dog podoplanin expressed in renal glomerulus by a novel monoclonal antibody PMab-38 in immunohistochemistry.

Downloaded by Yukinari Kato from www.liebertpub.com at 10/12/19. For personal use only.

EPITOPE OF PMAB-235

Monoclon Antib Immunodiagn Immunother 2016;35:
212–216.
26. Yamada S, Itai S, Nakamura T, Yanaka M, Saidoh N,
Chang YW, Handa S, Harada H, Kagawa Y, Ichii O,
Konnai S, Kaneko MK, and Kato Y: PMab-52: Specific andsensitive monoclonal antibody against cat podoplanin for
immunohistochemistry. Monoclon Antib Immunodiagn
Immunother 2017;36:224–230.
27. Honma R, Ogasawara S, Kaneko M, Fujii Y, Oki H, Nakamura T, Takagi M, Konnai S, and Kato Y: PMab-44 detects
bovine podoplanin in immunohistochemistry. Monoclon Antib Immunodiagn Immunother 2016;35:186–190.
28. Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, andKaneko MK: Anti-horse podoplanin monoclonal antibody
PMab-219 is useful for detecting lymphatic endothelial
cells by immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2018;37:272–274.
29. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T,
Yanaka M, Handa S, Mizuno T, Maeda K, Fukui M,
Harada H, Kaneko MK, and Kato Y: Establishment of
Monoclonal Antibody PMab-202 Against Horse Podoplanin. Monoclon Antib Immunodiagn Immunother 2018;37:
233–237.
30. Furusawa Y, Yamada S, Itai S, Nakamura T, Yanaka M,
Sano M, Harada H, Fukui M, Kaneko MK, and Kato Y:
PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin. Biochem Biophys
Rep 2019;18:100616.
31. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T,
Yanaka M, Fukui M, Harada H, Mizuno T, Sakai Y, Takasu
M, Kaneko MK, and Kato Y: PMab-210: A Monoclonal219

Antibody Against Pig Podoplanin Monoclon. Antib Immunodiagn Immunother 2019;38:30–36.
32. Kato Y, Yamada S, Furusawa Y, Itai S, Nakamura T, Yanaka M, Sano M, Harada H, Fukui M, and Kaneko MK:
PMab-213: A monoclonal antibody for immunohistochemical analysis against pig podoplanin Monoclon. AntibImmunodiagn Immunother 2019;38:18–24.
33. Sekiguchi T, Takemoto A, Takagi S, Takatori K, Sato S,
Takami M, and Fujita N: Targeting a novel domain in
podoplanin for inhibiting platelet-mediated tumor metastasis. Oncotarget 2015;7:3934–3946.
34. Kaneko MK, Nakamura T, Honma R, Ogasawara S, Fujii Y,
Abe S, Takagi M, Harada H, Suzuki H, Nishioka Y, and Kato
Y: Development and characterization of anti-glycopeptide
monoclonal antibodies against human podoplanin, using
glycan-deficient cell lines generated by CRISPR/Cas9 and
TALEN. Cancer Med 2017;6:382–396.

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: May 7, 2019
Accepted: July 11, 2019