MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 1, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0048

PMab-213:
A Monoclonal Antibody for Immunohistochemical
Analysis Against Pig Podoplanin

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Yukinari Kato,1,2 Shinji Yamada,2 Yoshikazu Furusawa,1–3 Shunsuke Itai,2,4 Takuro Nakamura,2Miyuki Yanaka,2 Masato Sano,2 Hiroyuki Harada,4 Masato Fukui,3 and Mika K. Kaneko2

Podoplanin (PDPN) is known to be expressed in normal tissues, including lymphatic endothelial cells, renal
podocytes, and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog,
cat, and bovine PDPN have already been established; however, mAbs against pig PDPN (pPDPN) are lacking.
In the present study, mice were immunized with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1
cells (CHO/pPDPN), and hybridomas producing mAbs against pPDPN were identified by flow cytometricscreening. One of the mAbs, PMab-213 (IgG2b, kappa), could specifically detect CHO/pPDPN cells throughflow cytometry and detect pPDPN through western blot analysis. KD of PMab-213 for CHO/pPDPN was
determined to be 2.1 · 10-9 M, indicating a high affinity for CHO/pPDPN. Furthermore, PMab-213 strongly
stained lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells through immunohistochemistry. PMab-213 is expected to be useful in investigating the function of pPDPN.
Keywords: pig podoplanin, PDPN, PMab-213

tion of pPDPN. In the present study, we immunized mice with
Chinese hamster ovary (CHO)/pPDPN cells and establishedhybridomas that could produce mAbs against pPDPN.

IntroductionP

odoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in normal tissues, including lymphatic endothelial cells, renal podocytes, and type I alveolar
cells of lung.(1,2) The expression of human PDPN has beenreported in several malignant tumors, including malignant
brain tumors,(3–6) oral squamous cell carcinomas,(7) esophageal cancers,(8) lung cancers,(9) malignant mesotheliomas,(10,11) osteosarcomas,(12–14) chondrosarcomas,(13) andtesticular tumors.(15) Moreover, the expression of PDPN is
associated with malignant progression and cancer metastasis.(3,16,17) The interaction between PDPN on lymphatic endothelial cells and C-type lectin-like receptor-2 on platelets
has been shown to facilitate embryonic blood/lymphatic
vessel separation.(1,16,18–24)
We have previously developed monoclonal antibodies(mAbs) against human,(25) mouse,(25) rat,(26) rabbit,(27) dog,(28)cat,(29) and bovine(30) PDPNs. However, mAbs against pig
PDPN (pPDPN), which are useful for immunohistochemical
analysis, are lacking. Specific and sensitive mAbs against
pPDPN are necessary to investigate the expression and func-

Materials and Methods
Cell lines

P3X63Ag8U.1 (P3U1) and CHO-K1 cells were obtained
from the American Type Culture Collection (Manassas, VA).
The pig kidney cell line PK-15 was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka,
Japan). pPDPN bearing an N-terminal PA16 tag (PA16pPDPN) was inserted into a pCAG-Ble vector (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan). The
PA16 tag consists of 16 amino acids (GLEGGVAMPGAEDDVV).(31) CHO-K1 cells were transfected with
pCAG-Ble/PA16-pPDPN using Lipofectamine LTX with
Plus Reagent (Thermo Fisher Scientific, Inc., Waltham, MA).
Stable transfectants were selected by limiting dilution andcultivated in a medium containing 0.5 mg/mL zeocin (InvivoGen, San Diego, CA).

1

New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
3
ZENOAQ RESOURCE CO., LTD., Koriyama, Japan.
4
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
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PMAB-213 AGAINST PIG PDPN

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FIG. 1. Schematic illustration of CBIS. Stable transfectants
expressing the protein of interest are used as an immunogenwith no purification procedure. The selection of hybridomas
secreting specific mAbs is performed through flow cytometry
using parental and transfectant cells. CBIS, Cell-Based Immunization and Screening; mAb, monoclonal antibody.
P3U1, CHO-K1, and CHO/pPDPN cells were cultured in
RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan),and PK-15 cells were cultured in Dulbecco’s Modified Eagle’s
medium (Nacalai Tesque, Inc.). All media were supplemented
with 10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc.), 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai Tesque, Inc.).
Cells were grown at 37C in a humidified environment with anatmosphere of 5% CO2 and 95% ambient air.
Animals

Female BALB/c mice (6 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). Animals were housed under
specific pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved all the animal
experiments described in this study. Microminipig (Fuji
Micra, Inc., Shizuoka, Japan) tissues were collected at autopsy at Gifu University and fixed in 4% paraformaldehyde.
Production of hybridomas

One BALB/c mouse was immunized against CHO/pPDPN
cells (1 · 108), which were administered intraperitoneally(i.p.) together with Imject Alum (Thermo Fisher Scientific,Inc.). The procedure included an additional three immunizations followed by a final booster injection administered i.p.
2 days before spleen cells were harvested—a total of fiveimmunizations. Subsequently, these spleen cells were fusedwith P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN) and the hybridomas were grown in RPMI
medium supplemented with hypoxanthine, aminopterin, andthymidine for selection (Thermo Fisher Scientific, Inc.). The
culture supernatants were screened using flow cytometry.

FIG. 2. Detection of pPDPN through flow cytometry using PMab-213. CHO/pPDPN, CHO-K1, and PK-15 cells
were treated with PMab-213 (red line) at a concentration of
10 mg/mL or control (gray) for 30 minutes followed by incubation with secondary antibodies. pPDPN, pig podoplanin; CHO, Chinese hamster ovary.
Flow cytometry

The cells were harvested following a brief exposure to
0.25% trypsin/1 mM ethylenediaminetetraaceticacid (Nacalai
Tesque, Inc.). The cells were washed with 0.1% bovine serum
albumin/phosphate-buffered saline (PBS) and treated withprimary mAbs for 30 minutes at 4C; thereafter, the cells weretreated with Alexa Fluor 488-conjugated anti-mouse immunoglobulin G (IgG, 1:2000; Cell Signaling Technology, Inc.,
Danvers, MA) or Oregon Green anti-rat IgG (1:2000; Thermo
Fisher Scientific, Inc.). Fluorescence data were collected using SA3800 Cell Analyzers (Sony Corp., Tokyo, Japan).
Western blot analysis

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.). The proteins were20

KATO ET AL.

subjected to electrophoresis on 5%–20% polyacrylamide gels
(Nacalai Tesque, Inc.) and then transferred onto a polyvinylidene difluoride membrane (Merck KGaA, Darmstadt,
Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.), each membrane was incubated with primarymAbs, such as 1 mg/mL of PMab-213, 1 mg/mL of anti-PA16
tag (NZ-1) or 1 mg/mL of anti-b-actin (AC-15; SigmaAldrich Corp., St. Louis, MO) and subsequently withperoxidase-conjugated anti-mouse IgG (1:1000; Agilent
Technologies, Santa Clara, CA) or anti-rat IgG (1:10,000;
Sigma-Aldrich Corp.). Bands were visualized with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation)using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

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Determination of binding affinity using flow cytometry

FIG. 3. Western blot analysis. Cell lysates of CHO-K1,
CHO/pPDPN, and PK-15 (10 mg) were electrophoresed andtransferred onto polyvinylidene difluoride membranes. The
membranes were incubated with l mg/mL of PMab-213,
1 mg/mL of anti-PA16 tag (NZ-1), or 1 mg/mL of anti-b-actin
and subsequently with peroxidase-conjugated anti-mouse or-rat IgG. IgG, immunoglobulin G.

CHO/pPDPN or PK-15 (2 · 105 cells) was suspended in
100 mL of serially diluted PMab-213, followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG (1:200;
Cell Signaling Technology, Inc.). Fluorescence data werecollected using EC800 Cell Analyzer (Sony Corp.). The
dissociation constant (KD) was obtained by fitting the binding
isotherms to built-in one-site binding models in GraphPad
PRISM 6 (GraphPad Software, Inc., La Jolla, CA).

FIG. 4. Immunohistochemical analyses using microminipig lungs. Histological sections of microminipig lung tissue wereincubated with 1 mg/mL of PMab-213 (A, B) or with blocking buffer (C, D), followed by an Envision+ Kit. Hematoxylin
and Eosin staining (E, F). Scale bar = 100 mm.

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PMAB-213 AGAINST PIG PDPN

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FIG. 5. Immunohistochemical analyses using microminipig kidneys. Histological sections of the microminipig kidney
tissue were incubated with 1 mg/mL of PMab-213 (A, B) or with blocking buffer (C, D), followed by an Envision+ Kit.
Hematoxylin and Eosin staining (E, F). Scale bar = 100 mm.
Immunohistochemical analyses

Tissues were processed to produce 4-mm paraffinembedded tissue sections, which were directly autoclaved incitrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo,
Japan) for 20 minutes. These tissue sections were blocked
using SuperBlock T20 (PBS) Blocking Buffer (Thermo
Fisher Scientific, Inc.) and incubated with PMab-213(1 mg/mL) for 1 hour at room temperature and treated using
the Envision+ Kit (Agilent Technologies, Inc.) for 30 minutes.
Color was developed using 3,3¢-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes, andcounterstaining was performed using Hematoxylin (FUJIFILM Wako Pure Chemical Corporation).
Results and Discussion

In the present study, we employed a Cell-Based Immunization and Screening (CBIS) method to develop specific andsensitive mAbs against pPDPN to facilitate the immunohistochemical analysis of paraffin-embedded tissue sections.
We have previously successfully utilized a CBIS method toestablish mAbs against various types of membrane proteins,

such as cat PDPN,(29) CD44,(31) CD133,(32) and PD-L1.(33)
One mouse was immunized with pPDPN-overexpressing
CHO-K1 (CHO/pPDPN) cells using an immunization andscreening procedure (Fig. 1). Hybridomas produced wereseeded into 96-well plates and cultivated for 10 days. Wells
positive for CHO/pPDPN and negative for CHO-K1 wereselected using flow cytometry. Moreover, PK-15 cells wereused to identify antibodies that reacted with the endogenous pPDPN. Screening identified strong signals against
CHO/pPDPN cells and weak or no signals against CHO-K1
cells in 54 out of 480 wells (11.3%). Of these 54 wells, 7
showed strong signal against PK-15 cells and were negative
against CHO-K1 cells. PMab-213 (IgG2b, kappa) was finallyidentified using limiting dilution from one of these wells.
PMab-213 recognized CHO/pPDPN but showed no reaction with CHO-K1 as assessed by flow cytometry (Fig. 2).
PMab-213 reacted with the PK-15 pig kidney cell line, indicating that PMab-213 was able to recognize pPDPN. On theother hand, PMab-213 showed no reaction with human,
mouse, rat, rabbit, dog, cat, or bovine PDPNs (Supplementary
Fig. S1). Furthermore, it showed no reaction with horse,
Tasmanian devil, tiger, alpaca, bear, goat, sheep, or whale

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KATO ET AL.

FIG. 6. Immunohistochemical analyses using microminipig colon. Histological sections of the microminipig colon tissue
were incubated with 1 mg/mL of PMab-213 (A, B) or with blocking buffer (C, D), followed by an Envision+ Kit. Hematoxylin and Eosin staining (E, F). Scale bar = 100 mm.

PDPNs (Supplementary Fig. S2), indicating that PMab-213 is
specific to pPDPN.
Western blot analysis performed using PMab-213 (Fig. 3)
demonstrated that PMab-213 detects pPDPN as a 40-kDa
band and a 25-kDa band in CHO/pPDPN cells. However,PMab-213 did not detect a 40-kDa band in PK-15 cells; thiscan be attributed to low expression levels of pPDPN in PK-15
cells or a low affinity of PMab-213 for PK-15 cells. A mAb
against PA16 tag (NZ-1) also detected a 40-kDa band and a
25-kDa band in CHO/pPDPN cells. The 40-kDa band is ahighly glycosylated form, whereas the 25-kDa band is an
unglycosylated form.(6,18)
Additionally, a kinetic analysis conducted using flow cytometry assessed the interaction of PMab-213 with CHO/pPDPN
and PK-15 cells. KD of PMab-213 for CHO/pPDPN and PK-15
cells was determined to be 2.1 · 10-9 M and 1.4 · 10-8 M,
respectively, indicating a high affinity for CHO/pPDPN anda moderate affinity for PK-15.
The immunohistochemical analyses revealed that PMab213 strongly stained pulmonary type I alveolar cells (Fig. 4),
renal podocytes and Bowman’s capsules (Fig. 5), and lym-

phatic endothelial cells of the colon (Fig. 6) obtained frommicrominipigs; these results indicate that PMab-213 is usefulfor the detection of pPDPN using immunohistochemistry. The
epitope of PMab-213 needs further investigation to clarify the
specificity and sensitivity of PMab-213 against pPDPN.
In conclusion, we have established an mAb against
pPDPN, PMab-213, which is applicable in flow cytometry,western blot, and immunohistochemical analyses. PMab-213
should prove useful for elucidating the pathophysiological
functions of pPDPN in future studies.
Acknowledgments

This research was supported in part by AMED under Grant
Numbers: JP18am0101078 (Y.K.), JP18am0301010 (Y.K.),
and JP18ae0101028 (Y.K.), and by JSPS KAKENHI Grant
Number 17K07299 (M.K.K.) and Grant Number 16K10748(Y.K.).
Author Disclosure Statement

No competing financial interests exist.

PMAB-213 AGAINST PIG PDPN
Supplementary Material

Supplementary Figure S1
Supplementary Figure S2

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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: November 20, 2018
Accepted: December 17, 2018