MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 3, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0005

Development of an Anti-Sheep Podoplanin Monoclonal
Antibody PMab-256 for Immunohistochemical
Analysis of Lymphatic Endothelial Cells

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Yukinari Kato,1,2 Yoshikazu Furusawa,1 Masato Sano,1 Junko Takei,1 Takuro Nakamura,1Miyuki Yanaka,1 Saki Okamoto,1 Saori Handa,1 Yu Komatsu,1 Teizo Asano,1
Yusuke Sayama,1 and Mika K. Kaneko1

Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses as a marker for lymphatic endothelial cells. We recently have developed anti-PDPN mAbs against
many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat,
horse, and bear. However, anti-sheep PDPN (sPDPN) has not yet been established. In this study, we used the CellBased Immunization and Screening method for the development of anti-sPDPN mAbs. RAP14 tag was added to
N-terminus of sPDPN, and anti-RAP14 tag mAb (PMab-2) was used to detect the expression level of sPDPN in flow
cytometry and western blot. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1(CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. One of the mAbs, PMab-256 (IgG1,
kappa), specifically detected CHO/sPDPN cells by flow cytometry and western blot. Furthermore, PMab-256
stained type I alveolar cells of lung, renal glomerulus and Bowman’s capsule, and lymphatic endothelial cells of lungand colon. Our findings suggest the potential usefulness of PMab-256 for the functional analyses of sPDPN.
Keywords: podoplanin, sheep, sPDPN, monoclonal antibody

Introduction

Materials and Methods
Cell lines

P

odoplanin (PDPN) is an O-glycosylated type I transmembrane protein expressed in renal podocytes, type I
alveolar cells of the lung, and lymphatic endothelial cellsof every organ.(1,2) It has platelet aggregation-stimulating(PLAG) domains(3) and a PLAG-like domain,(4–8) which bindto C-type lectin-like receptor-2 (CLEC-2).(9) The PDPN/
CLEC-2 interaction induces platelet aggregation and separation of embryonic blood/lymphatic vessels.(10) In morphological studies, PDPN is used as an important specificmarker of lymphatic vessels.(2)We previously developed monoclonal antibodies (mAbs)
against human,(11) mouse,(12) rat,(13) rabbit,(14) dog,(15) cat,(16)bovine,(17) pig,(18,19) Tasmanian devil,(20) alpaca,(21) tiger,(22)whale,(23) goat,(24,25) horse,(26,27) and bear(28,29) PDPNs using
the Cell-Based Immunization and Screening (CBIS) method.(30–32) These mAbs all demonstrated usefulness for flow
cytometry, western blot, and immunohistochemical analyses.
The purpose of the current study is to develop anti-sheep
PDPN (sPDPN) mAbs using the CBIS method to stain lymphatic endothelial cells through immunohistochemistry.
1
2

P3X63Ag8U.1 (P3U1) and Chinese hamster ovary (CHO)K1 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, United States). Synthesized
DNA (Eurofins Genomics KK, Tokyo, Japan) encoding sPDPN(accession no.: XM_004013802.4) plus an N-terminal RAP14
tag, which is recognized by an anti-RAP14 tag mAb (PMab-2),
was subcloned into a pCAG-Neo vector (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan). Plasmids were transfected using Lipofectamine LTX with Plus Reagent (Thermo
Fisher Scientific, Inc., Waltham, MA). Stable transfectants
were selected by limiting dilution and cultivation in medium
containing 0.5 mg/mL of G418 (Nacalai Tesque, Inc., Kyoto,
Japan).
The P3U1, CHO-K1, CHO/sPDPN,(33) CHO/human PDPN
(hPDPN),(3) CHO/mouse PDPN (mPDPN),(3) CHO/rat PDPN
(rPDPN),(13) CHO/rabbit PDPN (rabPDPN),(14) CHO/dog
PDPN (dPDPN),(15) CHO/bovine PDPN (bovPDPN),(17)CHO/cat PDPN (cPDPN),(16) CHO/pig PDPN (pPDPN),(19)CHO/horse PDPN (horPDPN),(26) CHO/tiger PDPN

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

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ANTI-SHEEP PDPN MAB(tigPDPN),(22) CHO/alpaca PDPN (aPDPN),(21) CHO/bear
PDPN (bPDPN),(19) CHO/Tasmanian devil PDPN
(tasPDPN),(19) CHO/goat PDPN (gPDPN),(22) and CHO/whale
PDPN (wPDPN)(23) were cultured in a Roswell Park Memorial
Institute (RPMI) 1640 medium (Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin,
100 lg/mL of streptomycin, and 25 lg/mL of amphotericin B(Nacalai Tesque, Inc.). Cells were grown in a humidified
incubator at 37C with atmosphere of 5% CO2 and 95% air.

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Western blot analysis

Cell lysates (10 lg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.). Proteins were thenelectrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred
onto polyvinylidene difluoride membranes (Merck KGaA,
Darmstadt, Germany). After blocking with 4% skim milk
(Nacalai Tesque, Inc.), membranes were incubated with
1 lg/mL of PMab-256, anti-RAP14 tag (clone: PMab-2),(13)or anti-b-actin (clone AC-15; Sigma-Aldrich Corp.,

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Hybridoma production

All animal experiments were performed according to relevant guidelines and regulations to minimize animal suffering and distress in the laboratory. Animal experiments wereapproved by the Animal Care and Use Committee of Tohoku
University (Permit number: 2016MdA-153). Mice weremonitored daily for health during the full 4-week duration of
the experiment. Body weight loss exceeding 25% of total
body weight was defined as a humane end point. Mice wereeuthanized by cervical dislocation, and death was verified byrespiratory and cardiac arrest.
Female BALB/c mice (6 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). The animals were housed under
specific pathogen-free conditions. To develop mAbs against
sPDPN, we used the CBIS method.(16,30–32) Briefly, three
BALB/c mice were immunized with CHO/sPDPN cells(1 · 108) by the intraperitoneal (i.p.) route together with the
Imject Alum (Thermo Fisher Scientific, Inc.). The procedureincluded three additional immunizations followed by a final
booster injection administered i.p. 2 days before the harvestof spleen cells. Harvested spleen cells were subsequently
fused with P3U1 cells using PEG1500 (Roche Diagnostics,
Indianapolis, IN), and the hybridomas were grown in an
RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific,Inc.). Culture supernatants were screened by flow cytometry.
Flow cytometry

Cells were harvested following a brief exposure to 0.25%
trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai
Tesque, Inc.). They were washed with 0.1% bovine serum
albumin in phosphate-buffered saline (PBS) and treated withprimary mAbs for 30 minutes at 4C. Thereafter, the cells
were treated with Alexa Fluor 488-conjugated anti-mouse
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 theSA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).
Determination of binding affinity by flow cytometry

CHO/sPDPN was suspended in 100 lL of serially diluted
PMab-256, and Alexa Fluor 488-conjugated anti-mouse IgG(1:200; Cell Signaling Technology, Inc.) was added. Fluorescence data were collected using the EC800 Cell Analyzer(Sony Corp.). The dissociation constant (KD) was calculated
by fitting the binding isotherms to built-in one-site binding
models in GraphPad PRISM 6 (GraphPad Software, Inc., La
Jolla, CA).

FIG. 1. Schematic illustration of the CBIS method. Stabletransfectants expressing sPDPN were used as an immunogen
without purification. Selection of hybridomas secreting antisPDPN mAbs was performed by flow cytometry using parental and transfectant cells. CBIS, Cell-Based Immunizationand Screening; mAbs, monoclonal antibodies; sPDPN, sheep
podoplanin.

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KATO ET AL.

FIG. 2. Detection of sPDPN by flow cytometry using PMab-256. (A) Schematic illustration of sPDPN. sPDPN possesses
a PLAG domain, PLD, and transmembrane domain. RAP14 was linked to the N-terminal of sPDPN. (B) CHO-K1 andCHO/sPDPN cells were treated with PMab-256 (red line) or anti-RAP14 tag (PMab-2; blue line) at a concentration of
1 mg/mL or 0.1% BSA in PBS (gray) for 30 minutes, followed by incubation with secondary antibodies. BSA, bovine serum
albumin; CHO, Chinese hamster ovary; PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain; PBS, phosphatebuffered saline.

St. Louis, MO), followed by incubation with peroxidaseconjugated anti-mouse immunoglobulin (diluted 1:2000;
Agilent Technologies, Inc., Santa Clara, CA). Membranes
were finally developed using ImmunoStar LD (FUJIFILM
Wako Pure Chemical Corporation) with a Sayaca-Imager(DRC Co. Ltd., Tokyo, Japan).
Immunohistochemical analyses

FIG. 3. Determination of binding affinity of PMab-256.
CHO/sPDPN was suspended in 100 mL of serially diluted
PMab-256. Alexa Fluor 488-conjugated anti-mouse IgG wasthen added. Fluorescence data were collected using theEC800 Cell Analyzer.

Normal sheep tissues were collected from two sheep after
autopsy at Hokkaido University, fixed in 10% neutralbuffered formalin, and processed routinely to make formalinfixed paraffin-embedded (FFPE) tissue sections as in ourprevious study.(33) Histological sections of 4 lm thickness
were directly autoclaved in citrate buffer (pH 6.0; Nichirei
Biosciences, Inc., Tokyo, Japan) for 20 minutes. After
blocking with SuperBlock T20 (PBS) Blocking Buffer(Thermo Fisher Scientific Inc.), sections were incubated with
PMab-256 (5 lg/mL) for 1 hour at room temperature andtreated using Envision+ Kit (Agilent Technologies, Inc.) for
30 minutes. Color was developed using 3,3-diaminobenzidine

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FIG. 4. Cross-reaction of PMab-256 with PDPNs of other species by flow cytometry. CHO-K1 cells transfected with PDPNsof other species were treated with PMab-256 (red line) (A) or each positive control (blue line) (B) at a concentration of 5 mg/mL
or 0.1% BSA in PBS (gray) for 30 minutes, followed by incubation with secondary antibodies. PDPN, podoplanin.
85

86

tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2
minutes, and counterstaining was performed with hematoxylin (FUJIFILM Wako Pure Chemical Corporation).
Results
Establishment of anti-sPDPN mAbs

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To develop anti-sPDPN mAbs, we used the CBIS method,in which stable transfectants are used for immunization, andscreening was performed using flow cytometry (Fig. 1). We
immunized three mice with CHO/sPDPN cells, which overexpress sPDPN (Fig. 2A). Resulting hybridomas were seeded
into 96-well plates, and CHO/sPDPN-positive and CHO-K1negative wells were selected. This screening approach identified strong signals from CHO/sPDPN and weak or no signals from CHO-K1 in 22 of the 1440 wells (1.5%). PMab-256(IgG1, kappa) was finally selected using western blotting andimmunohistochemistry.
Flow cytometric analyses

We performed flow cytometry using PMab-256 against
CHO/sPDPN and CHO-K1. PMab-256 recognized CHO/s
PDPN but not CHO-K1 (Fig. 2B). As expected, an anti-RAP14
tag mAb (PMab-2) also detected CHO/sPDPN. These results
indicate that PMab-256 is useful for detecting sPDPN in flow
cytometry analysis.

KATO ET AL.

We next measured the apparent binding affinity of PMab256 with CHO/sPDPN using flow cytometry. The KD of PMab256 for CHO/sPDPN was 6.9 · 10-9 M (Fig. 3), indicating that
PMab-256 possesses high affinity for CHO/sPDPN cells.
The cross-reaction of PMab-256 with the other PDPNs,
which are overexpressed in CHO-K1 cells, was then assessed. PMab-256 cross-reacted with hPDPN, bovPDPN,
gPDPN, aPDPN, tigPDPN, and wPDPN (Fig. 4A). Expression levels of these PDPNs were confirmed by each specificanti-PDPN mAb (Fig. 4B).
Western blot analyses

We next investigated whether PMab-256 can be applied towestern blot analysis using cell lysates of CHO-K1 or
CHO/sPDPN. As shown in Figure 5, PMab-256 detectedsPDPN as a 55-kDa band (N-terminal RAP14 tag + sPDPN)
in CHO/sPDPN cell lysates, but this antibody did not detect
any bands from CHO-K1 cells. Anti-RAP14 tag (PMab-2) asa positive control also detected a band of the same molecularweight from CHO/sPDPN. These results indicate that PMab256 is very useful for detecting sPDPN in western blot
analysis.
Immunohistochemical analyses

Finally, we investigated whether PMab-256 can be applied
to immunohistochemical analysis using FFPE sheep sections.
For this assessment, we selected normal tissues of sheep lung,

FIG. 5. Western blot analysis. Cell lysates of CHO-K1 and CHO/sPDPN (10 mg) were electrophoresed and transferred
onto PVDF membranes. The membranes were incubated with l mg/mL of PMab-256, anti-RAP14 tag (PMab-2), and anti-bactin and subsequently with peroxidase-conjugated anti-mouse immunoglobulin. PVDF, polyvinylidene difluoride.

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FIG. 6. Immunohistochemical analyses of the sheep lung. FFPE tissue sections of sheep lung were incubated with 5 mg/mL of
PMab-256 (A, B) or blocking buffer (C, D), followed by the Envision + Kit. (E, F) HE staining. Red and yellow arrows indicate
lymphatic endothelial cells. Scale bar = 100 mm. FFPE, Formalin-fixed paraffin-embedded; HE, hematoxylin and eosin.

FIG. 7. Immunohistochemical analyses of the sheep kidney. FFPE tissue sections of sheep kidneys were incubated with 5 mg/mL
of PMab-256 (A, B) or blocking buffer (C, D), followed by that with the Envision + Kit. (E, F) HE staining. Scale bar = 100 mm.
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KATO ET AL.

FIG. 8. Immunohistochemical analyses of the sheep colon. FFPE tissue sections of sheep colon were incubated with
5 mg/mL of PMab-256 (A, B) or blocking buffer (C, D), followed by the Envision + Kit. (E, F) HE staining. Red arrows
indicate lymphatic endothelial cells. Scale bar = 100 mm.
kidney, and colon, all of which have been reported to express
PDPN in other species, including human,(11) mouse,(12) rat,(13)rabbit,(14) dog,(15) cat,(16) bovine,(17) pig,(18,19) Tasmaniandevil,(20) alpaca,(21) tiger,(22) whale,(23) goat,(24,25) horse,(26,27)
and bear.(28,29) As depicted in Figure 6, PMab-256 strongly
stained type I alveolar cells within the lung and also clearlystained lymphatic endothelial cells of the lung (red arrow).
PMab-256 stained renal glomerulus and Bowman’s capsule(Fig. 7), although this staining pattern was not previously
observed by PMab-44 (an anti-bovPDPN mAb), which crossreacts with sPDPN.(33) The lymphatic endothelial cells (redarrow) of the colon were also clearly detected by PMab-256(Fig. 8), indicating that PMab-256 is very useful for detecting
lymphatic endothelial cells in FFPE tissues of sheep organs.
Discussion

Sheep possess anatomical and physiological structural
resemblance to humans(34) and have been used as experimental animals for many studies, including the immune
system,(35,36) development of the blood–brain barrier,(37)and lymphopoiesis/lymphocyte recirculation.(38) By contrast, detailed morphological studies of sheep lymphatic
vessels have not been performed due to the lack of specificmarkers for lymphatic endothelial cells.
In our previous study, we investigated possible crossreactions between anti-PDPN mAbs and sPDPN.(33) Flowcytometry analysis demonstrated that PMab-44 (an antibovPDPN mAb)(17) reacted with CHO/sPDPN. Other anti-

PDPN mAbs such as anti-mouse (PMab-1),(12) anti-rat(PMab-2),(13) anti-rabbit (PMab-32),(14) anti-dog (PMab-38
and PMab-48), and anti-cat (PMab-52)(16) did not react with
CHO/sPDPN, thus indicating then that only PMab-44 was
useful for the detection of sPDPN. We therefore investigatedthe expression of sPDPN using PMab-44 in sheep lungs using
immunohistochemical analyses. Sheep lungs were stained by
PMab-44 when EnVision FLEX Target Retrieval Solution
and a high pH were used for an antigen retrieval procedure,
although they were stained weakly using PMab-44 whencitrate buffer (pH 6) was used for antigen retrieval.(33) By
contrast, the renal glomerulus and Bowman’s capsule were
not stained by PMab-44. Furthermore, lymphatic endothelial
cells of the lung and colon were not stained by PMab-44.
Therefore, the development of novel anti-sPDPN mAbs,
which are applicable for detecting lymphatic endothelial cells
in immunohistochemical analysis, is crucial.
In this study, we used the CBIS method to develop novelanti-sPDPN mAbs (Fig. 1) because we have successfully
produced many mAbs against membrane proteins using the
CBIS method.(18–32) We immunized mice with CHO/sPDPN
cells and performed the first screening using only flow cytometry. Using this method abolishes the need for purified
protein for immunization and screening, which means that we
can develop mAbs more quickly compared with methods
using purified proteins and ELISA. The established clone
PMab-256 was characterized in this study, but two otherclones, PMab-253 and PMab-260, were established at thesame time. Both PMab-253 and PMab-260 were identified asDownloaded by Yukinari Kato from www.liebertpub.com at 06/24/20. For personal use only.

ANTI-SHEEP PDPN MAB

IgM class antibodies; and the sensitivities of PMab-253 andPMab-260 were less compared with PMab-256. For thesereasons, we selected PMab-256 (IgG1) for further analyses.
Although our previous study showed that PMab-44 stained
only type I alveolar cells and did not stain the lymphatic endothelial cells of sheep organs, PMab-256 strongly stained
lymphatic endothelial cells of the lung (Fig. 6) or colon (Fig. 8).
Our recent study showed that PMab-241, one of the antibPDPN mAbs, reacted with lymphatic endothelial cells, but notwith type I alveolar cells of the bear lung.(29) By contrast,
PMab-247, the other anti-bPDPN mAb, reacted with not only
lymphatic endothelial cells but also type I alveolar cells andrenal podocytes,(28) suggesting that the binding epitope of eachmAb is critical to distinguish PDPN-expressing lymphatic endothelial cells from other PDPN-expressing tissues. The binding epitope of PMab-256 should be determined in future studies
to clarify mechanisms for which sheep lymphatic endothelial
cells were strongly stained. PMab-256 may be advantageousfor detailed morphological studies of sheep lymphatic vessels.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by AMED underGrant Numbers: JP20am0401013 (Y.K.), JP20am0101078(Y.K.), and JP20ae0101028 (Y.K.) and by JSPS KAKENHI
Grant Number 17K07299 (M.K.K.) and Grant Number
19K07705 (Y.K.).
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi
Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: February 25, 2020
Accepted: March 29, 2020