MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 3, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0016

Short Communications

Characterization of the Anti-Bovine Podoplanin
Monoclonal Antibody PMab-44

Downloaded by Yukinari Kato from online.liebertpub.com at 06/21/17. For personal use only.

Shinji Yamada,1 Ryusuke Honma,2 Mika K. Kaneko,1 Takuro Nakamura,1 Miyuki Yanaka,1Noriko Saidoh,1 Michiaki Takagi,2 Satoru Konnai,3 and Yukinari Kato1,4

A type I transmembrane sialoglycoprotein podoplanin (PDPN) is expressed in several normal cells, including
podocytes of the kidney, type I alveolar cells of the lung, and lymphatic endothelial cells. We recently produced
an anti-bovine PDPN (bovPDPN) monoclonal antibody (mAb), PMab-44, by immunizing mice with recombinant proteins of bovPDPN. In this study, we determined the critical epitope of PMab-44 for the recognition of
bovPDPN using many deletion mutants and point mutants of bovPDPN. Flow cytometric analyses revealed thatthe epitope of PMab-44 was Glu46-Thr50, which corresponds to platelet aggregation-stimulating (PLAG)
domain-3. The important amino acids in the PMab-44 epitope were determined to be Glu46, Tyr48, and Thr50.
Western blot analysis also confirmed these results, indicating that the PLAG domain of bovPDPN is also
important in immunogenicity for producing useful anti-PDPN mAbs.
Keywords: podoplanin, bovine PDPN, PMab-44

Introduction

A

type I transmembrane sialoglycoprotein podoplanin(PDPN), also known as Aggrus,(1) activates platelet
aggregation by binding to the C-type lectin-like receptor-2(CLEC-2) on platelets.(2–4) The interaction between PDPN
and CLEC-2 was mainly observed at Glu47 and Asp48 in theplatelet aggregation-stimulating (PLAG) domain and thea2,6-linked sialic acid at Thr52 of human PDPN.(2,4,5) This
interaction facilitates blood/lymphatic vessel separation.(6)The actin cytoskeleton is rearranged in CLEC-2-expressing
dendritic cells to promote efficient motility along PDPNexpressing stromal surfaces.(7) The signal from CLEC-2 to
PDPN controls the contractility of fibroblastic reticular cells
and lymph node microarchitecture.(8) The physical elasticity
of lymph nodes is maintained by the PDPN of stromal fibroblastic reticular cells.(9)PDPN is expressed in several normal cells, including
lymphatic endothelial cells, renal podocytes, pulmonary typeI alveolar cells, osteocytes, and chondrocytes.(10) The expression of PDPN has been reported in many malignant tumors, such as oral squamous cell carcinomas,(11) malignant
brain tumors,(12–15) lung cancers,(16) esophageal cancers,(17)malignant mesotheliomas,(18,19) testicular tumors,(20) osteosarcomas,(10,21,22) and chondrosarcomas.(10) PDPN expres-sion is also associated with malignant progression and cancer
metastasis.(2,12,23)
We had previously established the anti-bovine PDPN
(bovPDPN) monoclonal antibody (mAb) PMab-44 (IgG1, kappa), which was useful for detecting podocytes and lymphatic
endothelial cells in normal bovine tissues in immunohistochemistry.(24) PMab-44 also detected bovPDPN specifically inflow cytometry. Our previous research revealed that bovPDPN
lacks platelet aggregation-inducing activity because the predicted amino acid sequence of bovPDPN has a sporadic deletionin the PLAG domain.(25) Therefore, the functional character of
bovPDPN may be somewhat different from other mammalian
PDPN. However, bovPDPN has so far attracted little attention
apart from a few studies.(26,27)
In this study, we further determined the epitope of PMab44 in flow cytometry and western blotting. This information
could be useful in bovPDPN veterinary research.
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 was purchased from theAmerican Type Culture Collection (ATCC, Manassas, VA).
CHO-K1 cells were transfected with the bovPDPN-MAP
tag(28) plasmid using a Gene Pulser Xcell electroporation

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Orthopedic Surgery, Yamagata University Faculty of Medicine, Yamagata, Japan.
Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
4
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
2
3

129

130

system (Bio-Rad Laboratories, Inc., Berkeley, CA). CHO-K1
and CHO/bovPDPN were cultured in RPMI 1640 medium(Nacalai Tesque, Inc., Kyoto, Japan), supplemented with
10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc., Waltham, MA), 100 units/mL of penicillin,
100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B(Nacalai Tesque, Inc.) at 37C in a humidified atmosphere of
5% CO2 and 95% air.

Downloaded by Yukinari Kato from online.liebertpub.com at 06/21/17. For personal use only.

Production of bovPDPN mutants

The amplified bovPDPN cDNA was subcloned into a
pCAG vector (Wako Pure Chemical Industries Ltd., Osaka,
Japan) and a MAP tag(28) was added at the N-terminus. Deletion mutation of a bovPDPN sequence was performed usinga HotStar HiFidelity polymerase chain reaction (PCR; Qiagen, Inc., Hilden, Germany) with oligonucleotides containing
the desired mutations. PCR fragments bearing the desired
mutations were inserted into a pCAG vector using the InFusion PCR Cloning Kit (Clontech, Palo Alto, CA). Substitutions of amino acids to alanine in bovPDPN sequenceswere conducted using the QuikChange lightning site-directedmutagenesis kit (Agilent Technologies, Inc., Santa Clara,
CA). CHO-K1 cells were transfected with the plasmids using
a Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Inc.).

YAMADA ET AL.
Flow cytometry

Cells were harvested by brief exposure to 0.25% trypsin/
1 mM EDTA (Nacalai Tesque, Inc.). After washing with
0.1% BSA/PBS, the cells were treated with PMab-44 (10 mg/mL) and anti-MAP tag(28) (clone: PMab-1, 1 mg/mL) for 30
minutes at 4C followed by treatment with Oregon greenconjugated anti-mouse IgG or anti-rat IgG (1:1000 diluted;
Thermo Fisher Scientific, Inc.). Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp., Tokyo,
Japan). PMab-1 was previously developed against mouse
PDPN(29) and has been utilized as an anti-MAP tag mAb.(28)PMab-1 does not crossreact with other PDPN proteins suchas bovPDPN,(24) dog PDPN,(30–32) rat PDPN,(33) rabbit
PDPN,(34,35) and human PDPN.(29)Western blot analyses

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate(SDS) sample buffer (Nacalai Tesque, Inc.). The proteins
were electrophoresed on 5%–20% polyacrylamide gels
(Wako Pure Chemical Industries Ltd.) and were transferred
onto a PVDF membrane (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque,Inc.), the membrane was incubated with PMab-44, antiMAP tag(28) (clone: PMab-1), and anti-b-actin (clone AC15; Sigma-Aldrich Corp., St. Louis, MO) and then with

FIG. 1. Epitope mapping of an anti-bovPDPN mAb PMab-44 using deletion mutants by flow cytometry. (A) Deletionmutants of bovPDPN (dN23, dN40, dN60, dN80, and dN100) were treated with PMab-44 (10 mg/mL) and PMab-1 (1 mg/mL) for 30 minutes at 4C followed by treatment with Oregon green-conjugated anti-mouse IgG or anti-rat IgG. (B)Deletion mutants of bovPDPN (dN23, dN45, dN50, and dN55) were treated with PMab-44 (10 mg/mL) and PMab-1 (1 mg/mL) for 30 minutes at 4C followed by treatment with Oregon green-conjugated anti-mouse IgG or anti-rat IgG. Fluorescence data were collected using a Cell Analyzer EC800. Red line: PMab-44 or anti-PA tag. Black line: negative control.
bovPDPN, bovine PDPN; mAb, monoclonal antibody; PDPN, podoplanin.

ANTI-BOVINE MAB PMAB-44

131

peroxidase-conjugated anti-mouse or anti-rat antibodies(1:1000 diluted; Dako; Agilent Technologies, Inc.). Subsequently, the membrane was developed with the Pierce
Western Blotting Substrate Plus (Thermo Fisher Scientific,Inc.) or the ImmunoStar LD Chemiluminescence Reagent(Wako Pure Chemical Industries Ltd.) using a SayacaImager (DRC Co. Ltd., Tokyo, Japan).

CHO/bovPDPN (2 · 105 cells) were resuspended with
100 mL of serially diluted antibodies (0.6 ng/mL–10 mg/mL)
followed by secondary anti-mouse IgG (Thermo Fisher Scientific, Inc.). Fluorescence data were collected using a cell
analyzer (EC800; Sony Corp.). The dissociation constants(KD) were obtained by fitting the binding isotherms using thebuilt-in one-site binding models in GraphPad PRISM 6(GraphPad Software, Inc., La Jolla, CA).
Results and Discussion
Epitope mapping of an anti-bovPDPN
mAb PMab-44 by flow cytometry

We previously showed the usefulness of an anti-bovPDPN
mAb PMab-44 in immunohistochemical analysis(24); in thisstudy we performed the epitope mapping using flow cytometry and western blot analysis.
First, we produced five N-terminal deletion mutants of
bovPDPN: dN23 (23–169 amino acids), dN40 (40–169 aminoacids), dN60 (60–169 amino acids), dN80 (80–169 aminoacids), and dN100 (100–169 amino acids). Flow cytometricanalysis revealed that PMab-44 reacted with dN23 and dN40; in
contrast, it did not react with dN60, dN80, and dN100 (Fig. 1A).
The anti-MAP tag PMab-1 recognized all deletion mutants.
These results indicate that the N-terminal of the PMab-44 epitope exists between Lys40 and Ala60 of bovPDPN.(25) Furthermore, we produced three N-terminal deletion mutants of
bovPDPN: dN45 (45–169 amino acids), dN50 (50–169 aminoacids), and dN55 (55–169 amino acids). Flow cytometricanalysis revealed that PMab-44 reacted with dN23 and dN45; incontrast, it did not react with dN50 and dN55 (Fig. 1B). The
anti-MAP tag PMab-1 recognized all deletion mutants. These
results indicate that the N-terminal of the PMab-44 epitope
exists between Val45 and Thr50 of bovPDPN.(25)
Next, we produced 10 point mutants of bovPDPN: V45A,
E46A, D47A, Y48A, T49A, T50A, T51A, P52A, A53G, andA54G. Figure 2 shows that two point mutants, such as E46A
andY48A, completely lost the reaction by PMab-44, and
T50A showed little reaction by PMab-44. All point mutants
were recognized by the anti-MAP tag PMab-1. These results‰

Downloaded by Yukinari Kato from online.liebertpub.com at 06/21/17. For personal use only.

Determination of the binding affinity
using flow cytometry

FIG. 2. Epitope mapping of an anti-bovPDPN mAb
PMab-44 using point mutants by flow cytometry. Point
mutants of bovPDPN (V45A, E46A, D47A, Y48A, T49A,
T50A, T51A, P52A, A53G, and A54G) were treated with
PMab-44 (10 mg/mL) and anti-MAP tag (1 mg/mL) for 30
minutes at 4C followed by treatment with Oregon greenconjugated anti-mouse IgG or anti-rat IgG. Fluorescencedata were collected using a Cell Analyzer EC800. Red line:
PMab-44 or anti-PA tag. Black line: negative control.

132

YAMADA ET AL.

indicate that three amino acids of bovPDPN, Glu46, Tyr48,
and Thr50, are critical for PMab-44 recognition.

Downloaded by Yukinari Kato from online.liebertpub.com at 06/21/17. For personal use only.

Epitope mapping of an anti-bovPDPN mAb PMab-44
by western blot analysis

We next performed western blot analysis to confirm theresults of epitope mapping of PMab-44 by flow cytometry. As
shown in Figure 3A, PMab-44 did not react with V45A,
E46A, D47A, Y48A, T49A, and T50A of bovPDPN. In
contrast, PMab-44 reacted with T51A, P52A, A53G, andA54G. All point mutants were detected by the anti-MAP tag
PMab-1. These results indicated that Val45, Glu46, Asp47,
Tyr48, Thr49, and Thr50 are important in the recognition ofPMab-44 against bovPDPN in western blot analysis. The
epitope of PMab-44 is illustrated in Figure 3B. The sequenceof the PMab-44 epitope (VEDYTT) shows low homology
with the corresponding peptide sequence of human PDPN
(AEDDVV), indicating that PMab-44 does not react withhuman PDPN.(24)
Determination of the binding affinity
using flow cytometry

We next performed a kinetic analysis of the interaction ofPMab-44 with CHO/bovPDPN using flow cytometry. As

FIG. 4. Determination of binding affinity using flow cytometry. CHO/bovPDPN cells were resuspended with 100 mLof serially diluted antibodies (0.6 ng/mL–10 mg/mL) followed
by secondary anti-mouse IgG. Fluorescence data were collected using a cell analyzer. CHO, Chinese hamster ovary.

shown in Figure 4, the KD of PMab-44 was determined as
3.3 · 10-9 M, indicating that PMab-44 possesses a high affinity against bovPDPN.
In conclusion, the most important amino acids for PMab44 recognition against bovPDPN were determined to be
Glu46, Tyr48, and Thr50; second in importance were Val45,
Asp47, and Thr49. Three amino acids, Glu46, Tyr48, andThr50, exist in the PLAG3 of bovPDPN,(25) indicating that
the PLAG3 of bovPDPN is also important for immunogenicity in producing useful anti-PDPN mAbs.
Acknowledgments

FIG. 3. Epitope mapping of an anti-bovPDPN mAb
PMab-44 using point mutants by western blot analysis. (A)Cell lysate (10 mg) was electrophoresed on 5%–20% polyacrylamide gels and transferred onto a PVDF membrane.
After blocking, the membrane was incubated with 1 mg/mL
of mAbs (PMab-44, anti-MAP tag, and anti-b-actin) andthen with peroxidase-conjugated secondary antibodies; themembrane was developed with Pierce Western Blotting
Substrate Plus or ImmunoStar LD Chemiluminescence Reagent and detected using a Sayaca-Imager. (B) Schematic
illustration of the epitope of PMab-44. Gray shade: identicalamino acid residue between bovPDPN and human PDPN.
Arrow head: conserved Thr of PLAG domain. Red character: PMab-44 epitope. PLAG domain, platelet aggregationstimulating domain.

This research was supported in part by grants from the
Science and Technology Research Promotion Program for
Agriculture, Forestry, Fisheries, and Food Industry, Japan(grant 26058B to S.K.), and the NARO, Bio-oriented Technology Research Advancement Institution (the special scheme
project on regional developing strategy: grant 16817557 toS.K.). This work was also supported in part by Project for
utilizing glycans in the development of innovative drug discovery technologies from Japan Agency for Medical Researchand Development, AMED (Y.K.), by the Basic Science andPlatform Technology Program for Innovative Biological
Medicine from AMED (Y.K.), by the Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) from
AMED (Y.K.), by the Regional Innovation Strategy Support
Program from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) of Japan (Y.K.), by JSPS
KAKENHI Grant Number 26440019 and 17K07299 (M.K.K.)
and 16K10748 (Y.K.). This work was performed in part under
the Cooperative Research Program of Institute for Protein
Research, Osaka University, CR-16-05 and CR-17-05 and by
the Grant for Joint Research Project of the Institute of Medical
Science, the University of Tokyo. The authors would like tothank Enago (www.enago.jp) for the English language review.
Author Disclosure Statement

No competing financial interests exist.

ANTI-BOVINE MAB PMAB-44

Downloaded by Yukinari Kato from online.liebertpub.com at 06/21/17. For personal use only.

References

1. 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.
2. 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.
3. 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.
4. 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 podoplaninmediated platelet activation, by cancer cells. J Biol Chem
2007;282:25993–26001.
5. Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK,
Kato Y, and Yamaguchi Y: A platform of C-type lectin-likereceptor CLEC-2 for binding O-glycosylated podoplanin
and nonglycosylated rhodocytin. Structure 2014;22:1711–
1721.
6. 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.
7. Acton SE, Astarita JL, Malhotra D, Lukacs-Kornek V,
Franz B, Hess PR, Jakus Z, Kuligowski M, Fletcher AL,
Elpek KG, Bellemare-Pelletier A, Sceats L, Reynoso ED,
Gonzalez SF, Graham DB, Chang J, Peters A, Woodruff M,
Kim YA, Swat W, Morita T, Kuchroo V, Carroll MC, Kahn
ML, Wucherpfennig KW, and Turley SJ: Podoplanin-richstromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2. Immunity
2012;37:276–289.
8. Astarita JL, Cremasco V, Fu J, Darnell MC, Peck JR,
Nieves-Bonilla JM, Song K, Kondo Y, Woodruff MC,
Gogineni A, Onder L, Ludewig B, Weimer RM, Carroll
MC, Mooney DJ, Xia L, and Turley SJ: The CLEC-2podoplanin axis controls the contractility of fibroblastic
reticular cells and lymph node microarchitecture. Nat Immunol 2015;16:75–84.
9. Acton SE, Farrugia AJ, Astarita JL, Mourao-Sa D, Jenkins
RP, Nye E, Hooper S, van Blijswijk J, Rogers NC, Snelgrove KJ, Rosewell I, Moita LF, Stamp G, Turley SJ, Sahai
E, and Reis e Sousa C: Dendritic cells control fibroblastic
reticular network tension and lymph node expansion. Nature 2014;514:498–502.
10. Ariizumi T, Ogose A, Kawashima H, Hotta T, Li G, Xu Y,
Umezu H, Sugai M, and Endo N: Expression of podoplaninin human bone and bone tumors: New marker of osteogenic and chondrogenic bone tumors. Pathol Int 2010;60:
193–202.
11. 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

133

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.
12. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T,
and Matsutani M: Increased expression of podoplanin inmalignant astrocytic tumors as a novel molecular marker ofmalignant progression. Acta Neuropathol (Berl) 2006;111:
483–488.
13. Mishima K, Kato Y, Kaneko MK, Nakazawa Y, Kunita A,
Fujita N, Tsuruo T, Nishikawa R, Hirose T, and Matsutani
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.
14. 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.
15. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
16. 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.
17. Schoppmann SF, Jesch B, Riegler MF, Maroske F,
Schwameis K, Jomrich G, and Birner P: Podoplanin expressing cancer associated fibroblasts are associated withunfavourable prognosis in adenocarcinoma of the esophagus. Clin Exp Metastasis 2013;30:441–446.
18. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
19. 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.
20. Kato Y, Sasagawa I, Kaneko M, Osawa M, Fujita N, andTsuruo T: Aggrus: A diagnostic marker that distinguishes
seminoma from embryonal carcinoma in testicular germ
cell tumors. Oncogene 2004;23:8552–8556.
21. Kaneko MK, Oki H, Ogasawara S, Takagi M, and Kato Y:
Anti-podoplanin monoclonal antibody LpMab-7 detects
metastatic legions of osteosarcoma. Monoclon Antib Immunodiagn Immunother 2015;34:154–161.
22. Kunita A, Kashima TG, Ohazama A, Grigoriadis AE, andFukayama M: Podoplanin is regulated by AP-1 and promotes platelet aggregation and cell migration in osteosarcoma. Am J Pathol 2011;179:1041–1049.
23. 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.
24. 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.
25. Kaneko MK, Kato Y, Kitano T, and Osawa M: Conservation of a platelet activating domain of Aggrus/podoplaninDownloaded by Yukinari Kato from online.liebertpub.com at 06/21/17. For personal use only.

134

as a platelet aggregation-inducing factor. Gene 2006;378:
52–57.
26. Gao Y, Qin L, Yang Y, Dong X, Zhao Z, Zhang G, andZhao Z: PDPN gene promotes the proliferation of immature
Bovine Sertoli cells in vitro. Anim Reprod Sci 2017;179:
35–43.
27. Nitta A, Shirasuna K, Nibuno S, Bollwein H, Shimizu T,
and Miyamoto A: Downregulation of lymphatic vessel
formation factors in PGF2alpha-induced luteolysis in thecow. J Reprod Dev 2013;59:296–301.
28. Fujii Y, Kaneko MK, and Kato Y: MAP Tag: A Novel
Tagging System for Protein Purification and Detection.
Monoclon Antib Immunodiagn Immunother 2016;35:293–299.
29. 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.
30. 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. Monoclon AntibImmunodiagn Immunother 2016;35:212–216.
31. Kaneko MK, Honma R, Ogasawara S, Fujii Y, Nakamura
T, Saidoh N, Takagi M, Kagawa Y, Konnai S, and Kato Y:
PMab-38 recognizes canine podoplanin of squamous cell
carcinomas. Monoclon Antib Immunodiagn Immunother2016;35:263–266.
32. Ogasawara S, Honma R, Kaneko MK, Fujii Y, Kagawa Y,
Konnai S, and Kato Y: Podoplanin expression in canine
melanoma. Monoclon Antib Immunodiagn Immunother2016;35:304–306.

YAMADA ET AL.

33. 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.
34. 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.
35. Honma R, Fujii Y, Ogasawara S, Oki H, Konnai S, Kagawa
Y, Takagi M, Kaneko MK, and Kato Y: Critical epitope ofanti-rabbit podoplanin monoclonal antibodies for immunohistochemical analysis monoclon. Antib Immunodiagn
Immunother 2016;35:65–72.

Address correspondence to:
Dr. Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: March 9, 2017
Accepted: April 16, 2017