MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume XX, Number XX, 2016ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2016.0022

Original Article

Specific Detection of Dog Podoplanin Expressed
in Renal Glomerulus by a Novel Monoclonal
Antibody PMab-38 in Immunohistochemistry
Ryusuke Honma,1,2 Mika K. Kaneko,1 Satoshi Ogasawara,1 Yuki Fujii,1
Satoru Konnai,3 Michiaki Takagi,2 and Yukinari Kato1

Podoplanin (PDPN) is expressed in several normal tissues including podocytes of renal glomerulus, lymphatic
endothelial cells (LECs), and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to Ctype lectin-like receptor-2 (CLEC-2) on platelet. Many monoclonal antibodies (mAbs) against human PDPN,
mouse PDPN, rat PDPN, rabbit PDPN, and bovine PDPN have been established, antidog PDPN (dPDPN) mAbs
have not been developed. Herein, we immunized mice with the recombinant proteins of dPDPN and developed
anti-dPDPN mAbs. One of the clones, PMab-38, is useful for detecting podocytes in immunohistochemical
analysis; in contrast, it did not react with LECs or type I alveolar cells. PMab-38 also detected dPDPN
specifically in flow cytometry and Western blot analysis. PMab-38 is expected to be useful for investigating thefunction of dPDPN, which is expressed in podocytes.
Manassas, VA). CHO-K1, stable CHO transfectants, andP3U1 were cultured in RPMI 1640 medium (Nacalai Tesque,Inc., Kyoto, Japan) supplemented with 10% heat-inactivatedfetal bovine serum (Thermo Fisher Scientific, Inc., Waltham,MA), 100 U/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.
Female BALB/c mice (4 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). Normal dog tissues were obtained from North lab (Hokkaido, Japan). Animals werehoused under pathogen-free conditions. The Animal Careand Use Committee of Tohoku University approved the animal experiments described herein.

Introduction

P

(1–4)

odoplanin (PDPN), also known as Aggrus/T1a,
is a type I transmembrane sialoglycoprotein. Dog PDPN
(dPDPN) was first reported as gp40.(5,6) PDPN is expressed inlymphatic endothelial cells (LECs), renal podocytes, pulmonary type I alveolar cells, osteocytes, and chondrocytes.(7,8)
The expression of human PDPN (hPDPN) has been reported
in many malignant tumors such as oral squamous cell carcinomas,(9) malignant brain tumors,(10–13) lung cancers,(14)esophageal cancers,(15) malignant mesotheliomas,(16,17) testicular tumors,(18) osteosarcomas,(8,19,20) and chondrosarcomas.(8)PDPN expression is also associated with malignant progression and cancer metastasis.(10,21,22)
PDPN activates platelet aggregation by binding to C-type
lectin-like receptor-2 (CLEC-2) on platelet.(21,23–25) The interaction between PDPN and CLEC-2 facilitates blood/
lymphatic vessel separation.(26) PDPN is also expressed inhuman fetal rib and chondrocytes of the proliferative andhypertrophic regions of the growth plate.(27)
Herein, we immunized mice with the recombinant proteinsof dPDPN and established anti-dPDPN monoclonal antibodies(mAbs).

Hybridoma production

The dPDPN with N-terminal PA tag and C-terminal
RAP tag-MAP tag (PA-dPDPN-RAP-MAP) was inserted
into pCAG-zeo vector (Wako Pure Chemical Industries
Ltd., Osaka, Japan). RAP tag consists of 12 amino acids(DMVNPGLEDRIE) and MAP tag consists of 12 aminoacids (GDGMVPPGIEDK). CHO-K1 was transfected with
pCAG-zeo/PA-dPDPN-RAP-MAP using Gene Pulser Xcell
electroporation system (Bio-Rad Laboratories, Inc., Berkeley, CA). For the purification of PA-dPDPN-RAP-MAP fromcell membrane, we used the PA tag system.(28,29) BALB/cmice were immunized by intraperitoneal (i.p.) injection of
100 mg of recombinant PA-dPDPN-RAP-MAP together with

Materials and Methods
Cell lines, dog tissues, and animals

Chinese hamster ovary (CHO)-K1 and P3U1 were purchased from the American Type Culture Collection (ATCC,
1
2
3

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, Yamagata, Japan.
Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.

1

2

Imject Alum (Thermo Fisher Scientific, Inc.). After several
additional immunizations of 50 mg, a booster injection of
100 mg was given i.p. 2 days before spleen cells were harvested. The spleen cells were fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in RPMI medium with hypoxanthine,aminopterin, and thymidine selection medium supplement(Thermo Fisher Scientific, Inc.).
The culture supernatants were screened using enzymelinked immunosorbent assay (ELISA) for binding to purified
recombinant PA-dPDPN-RAP-MAP. Recombinant proteinsof PA-dPDPN-RAP-MAP were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.)
at 1 mg/mL for 30 minutes. After blocking with 1% BSA in
0.05% Tween20/phosphate-buffered saline (PBS; Nacalai
Tesque, Inc.), the plates were incubated with culture supernatant followed by 1:3000 diluted peroxidase-conjugatedantimouse IgG (Dako; Agilent Technologies, Inc., Santa
Clara, CA). The enzymatic reaction was conducted with a 1Step Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). The
optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc.).

HONMA ET AL.

Aldrich Corp., St. Louis, MO) and then with peroxidaseconjugated antimouse IgG (1:1000 diluted; Dako) and developed with the Pierce Western Blotting Substrate Plus
(Thermo Fisher Scientific, Inc.) using a Sayaca-Imager (DRC
Co. Ltd., Tokyo, Japan).
Results
Production of mAbs against dPDPN

We immunized mice with the recombinant proteins, whichwere purified from CHO/PA-dPDPN-RAP-MAP cells, andthe ELISA screening was performed. Anti-PA tag mAb(clone: NZ-1), anti-MAP tag mAb (clone: PMab-1), and antiRAP tag mAb (clone: PMab-2) detected the PA-dPDPNRAP-MAP protein in Western blot analysis (data notshown). Among ELISA-positive wells, second screening was

Immunohistochemical analyses

Four-micrometer-thick histologic sections were deparaffinized in xylene and rehydrated and were autoclaved incitrate buffer (pH 6.0; Dako) for 20 minutes. Sections wereincubated with 10 mg/mL of primary mAbs for 1 hour atroom temperature followed by treatment with Envision+ kit
for 30 minutes (Dako). Color was developed using 3,3diaminobenzidine tetrahydrochloride (DAB; Dako) for 1
minute, and then the sections were counterstained with hematoxylin (Wako Pure Chemical Industries Ltd.).
Flow cytometry

Stable transfectant of CHO/PA-dPDPN-RAP-MAP wasestablished using SH800 (Sony Corp., Tokyo, Japan). The
other stable transfectants [CHO/PA-bovine PDPN (bovPDPN)RAP-MAP, CHO/hPDPN-FLAG, CHO/mouse PDPN
(mPDPN)-FLAG, CHO/rat PDPN (rPDPN)-His, and CHO/PArabbit PDPN (rabPDPN)] were previously established.(1,30–32)
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 primary mAbs
(1 mg/mL) for 30 minutes at 4C followed by treatment with
Oregon green-conjugated antimouse IgG (1:1000 diluted;
Thermo Fisher Scientific, Inc.). Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp.).
Western blot analysis

Cell lysates (10 mg) of CHO/PA-dPDPN-RAP-MAP, CHO/
bovPDPN-FLAG, CHO/hPDPN-FLAG, CHO/mPDPNFLAG, CHO/rPDPN-His, CHO/PA-rabPDPN, and CHO-K1
were boiled in SDS sample buffer (Nacalai Tesque, Inc.). The
proteins were electrophoresed on 5%–20% polyacrylamidegels (Wako Pure Chemical Industries Ltd.) and were transferred onto a polyvinylidene difluoride (PVDF) membrane(EMD Millipore Corp., Billerica, MA). After blocking with
4% skim milk (Nacalai Tesque, Inc.), the membrane was incubated with PMab-38 and anti-b-actin (clone AC-15; Sigma-

FIG. 1. Immunohistochemical analysis by PMab-38. Sections of dog kidney (A), dog colon (B), and dog lung (C) wereautoclaved in citrate buffer (pH 6.0). After blocking, they wereincubated with 10 mg/mL of PMab-38, followed by EnVision+
kit, and color was developed using DAB and counterstained withhematoxylin. PBS was used as negative control. HE staining was
performed against serial sections. Arrow head, renal glomerulus.
Scale bar: 100 mm. HE, hematoxylin and eosin.

MABS AGAINST DOG PODOPLANIN

performed by flow cytometry. Finally, positive wells wereselected in immunohistochemical analysis. After limiting
dilution, one of the clones, PMab-38 (IgG1, kappa), was established. PMab-38 reacted with podocytes of renal glomerulus (Fig. 1A), whereas it did not react with other normal

3cells such as LECs (Fig. 1B) or type I alveolar cells of lung(Fig. 1C), in which PDPN expression has been reported inother species.(3,7) These data indicate that PMab-38 is usefulfor immunohistochemistry using paraffin-embedded tissues.
Specificity of PMab-38 against dPDPN in flow
cytometry and Western blot

PMab-38 reacted with CHO/PA-dPDPN-RAP-MAP, notwith CHO/PA-bovPDPN-RAP-MAP, CHO/hPDPN-FLAG,
CHO/mPDPN-FLAG, CHO/rPDPN-His, CHO/PA-rabPDPN,
and CHO-K1 cells in flow cytometry (Fig. 2A). In Western
blot analysis, PMab-38 recognized only dPDPN of CHO/PAdPDPN-RAP-MAP, and did not react with PDPNs of CHO/
bovPDPN-FLAG, CHO/hPDPN-FLAG, CHO/mPDPN-FLAG,
CHO/rPDPN-His, CHO/PA-rabPDPN, or CHO-K1 cells(Fig. 2B). These results indicate that PMab-38 detects dPDPN
specifically.
Discussion

Although many mAbs and polyclonal Abs against PDPN
have been produced, anti-dPDPN mAbs have not been developed. Our previous reports revealed that PMab-44 (antibovPDPN mAb; mouse IgG1),(32) LpMab-12 (anti-hPDPN
mAb; mouse IgG1),(33) PMab-1 (anti-mPDPN mAb; rat
IgG2a),(34) PMab-2 (anti-rPDPN mAb; mouse IgG1),(30) and
PMab-32 (anti-rabPDPN mAb; mouse IgG1)(31,35) specifically react with CHO/bovPDPN, CHO/hPDPN, CHO/
mPDPN, CHO/rPDPN, and CHO/rabPDPN, respectively.
Many other anti-hPDPN mAbs also detect hPDPN specifically.(12,13,17,19,36–47) Herein, we immunized mice with therecombinant proteins of dPDPN, and developed several antidPDPN mAbs including PMab-38. When PMab-38 was incubated for 18 hours at 4C in immunohistochemistry, thestronger staining was observed in renal glomerulus, andBowman’s capsule was also stained by PMab-38, whereas
LECs or type I alveolar cells were not stained (data not shown).
These data demonstrate that PMab-38 is useful for immunohistochemistry using paraffin-embedded tissues, but the reaction of PMab-38 is limited to podocytes of renal glomerulus.
Taken together, PMab-38 could be useful to uncover thepathophysiological function of dPDPN in podocytes of renal
glomerulus. In the future, we should determine the epitope ofPMab-38 and clarify the reason for the specificity of PMab38 against podocytes of renal glomerulus in immunohistochemistry.
Acknowledgments

FIG. 2. Flow cytometric and Western blot analyses by PMab38. (A) Cells were treated with PMab-38 followed by treatment
with Oregon green-conjugated antimouse IgG. Black line:
negative control. (B) Cell lysates (10 mg) were electrophoresed
and transferred onto a PVDF membrane. After blocking, themembrane was incubated with 1 mg/mL of mAbs (PMab-38 or
anti-b-actin), and then with peroxidase-conjugated antimouse
IgG; the membrane was developed with Pierce Western Blotting Substrate Plus and detected using a Sayaca-Imager. mAbs,
monoclonal antibodies; PVDF, polyvinylidene difluoride.

We thank Takuro Nakamura, Noriko Saidoh, and Kanae
Yoshida for their excellent technical assistance. This work
was supported in part 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 (M.K.K.) and
Grant Number 25462242 and 16K10748 (Y.K.), by Takeda
Science Foundation (S.O.), by the Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) fromJapan Agency for Medical Research and Development,
AMED (Y.K.), and by the Basic Science and Platform
Technology Program for Innovative Biological Medicine
from AMED (Y.K.). This work was performed, in part, under

4

HONMA ET AL.

the Cooperative Research Program of Institute for Protein
Research, Osaka University, CR-15-05 and CR-16-05.
Author Disclosure Statement

No competing financial interests exist.
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Address correspondence to:Prof. Yukinari Kato
Department of Regional Innovation
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: May 12, 2016
Accepted: June 3, 2016