MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 34, Number 6, 2015ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2015.0041

Development of Sensitive Monoclonal Antibody
PMab-2 Against Rat Podoplanin
Hiroharu Oki,1,2* Ryusuke Honma,1,2,* Satoshi Ogasawara,1,* Yuki Fujii,1 Xing Liu,1,2Michiaki Takagi,2 Mika K. Kaneko,1 and Yukinari Kato1

Podoplanin (PDPN) is a platelet aggregation-inducing factor, which is known as an endogenous ligand of Ctype lectin-like receptor-2 (CLEC-2). PDPN is also expressed in several normal tissues, such as lung type I
alveolar cells and kidney podocytes. Although many monoclonal antibodies (MAbs) against human PDPN
(hPDPN) or mouse PDPN (mPDPN) have been established, anti-rat PDPN (rPDPN) MAbs, especially against
platelet aggregation-stimulating (PLAG) domain (29-54 amino acids) of rPDPN, have not been developed.
Therefore, functional analysis of rPDPN in normal tissues has been limited. Here, we immunized mice with
rPDPN peptides (38-51 amino acids) and developed a novel mouse anti-rPDPN MAb, PMab-2 (IgG1, kappa),
which possesses high affinity compared with anti-hPDPN or mPDPN MAbs. The KD of PMab-2 was determined
to be 5.9 · 10-10 M. PMab-2 is useful, not only in flow cytometry and Western blot analysis against endogenousrPDPN, which is expressed in rat dermal fibroblast, but also in immunohistochemistry against normal tissues.
PMab-2 showed extraordinarily high sensitivity in immunohistochemistry, indicating that PMab-2 is very
advantageous for functional analysis of rPDPN.

IntroductionP

odoplanin (PDPN/PA2.26/T1a/Aggrus) is a type I
transmembrane O-glycoprotein that possesses platelet
aggregating activity.(1–4) We recently performed crystallographic studies of human PDPN (hPDPN) in complex with Ctype lectin-like receptor-2 (CLEC-2).(5) The interaction with
CLEC-2 was mainly observed at Glu47 and Asp48 in theplatelet aggregation-stimulating (PLAG) domain of hPDPN(29-54 amino acids) and the a2-6 linked sialic acid at Thr52
of hPDPN. The platelet aggregation via PDPN-CLEC-2 interaction is critical for embryonic blood-lymphatic vascularseparation.(6) In adult tissues, PDPN is very useful for detecting lymphatic endothelial cells.(7) PDPN also possesses
many important functions. Interestingly, both PDPN-CLEC2 axis and CLEC-2-PDPN axis have been reported. PDPN
binds to CLEC-2 and rearranges the actin cytoskeleton in
dendritic cells to promote efficient motility along stromal
surfaces.(8) The development of ectopic lymphoid follicles
is dependent on Th17-expressing PDPN.(9) The signal from
CLEC-2 to PDPN controls the contractility of fibroblastic
reticular cells and lymph node microarchitecture.(10) The
physical elasticity of lymph nodes is maintained by PDPN
of stromal fibroblastic reticular cells.(11) Expression of

PDPN has also been reported in many cancers, including oral
cancers, malignant brain tumors, esophageal cancers, lungcancers, malignant mesotheliomas, testicular tumors, and osteosarcomas.(3,12–23) Moreover, PDPN expression in cancerassociated fibroblasts (CAFs) is involved in poor prognosis ofcancers.(24–28) PDPN is also reported in advanced atherosclerotic
lesions of human aortas.(29) Although anti-PDPN MAbs withhigh sensitivity and specificity are necessary to investigate thephysiological function of PDPN in normal tissues, almost all
anti-PDPN MAbs have been developed against hPDPN(17,30–39)
and mouse PDPN (mPDPN),(40–42) not against rat PDPN
(rPDPN).(1) Therefore, we immunized mice with PLAG domain
of rPDPN and developed high-sensitive MAbs against rPDPN.
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 and P3U1 were purchased from the American Type Culture Collection (ATCC,
Manassas, VA). Rat dermal fibroblast was obtained from Cell
Applications (San Diego, CA). CHO-K1 was transfected with
rPDPN, hPDPN, or mPDPN plasmids (CHO/rPDPN, CHO/
hPDPN, CHO/mPDPN) using Lipofectamine 2000 (Thermo
Fisher Scientific, Waltham, MA).(3) CHO-K1, CHO/rPDPN,

1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi, Japan.
Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, Yamagata, Japan.
*These authors contributed equally to this work.

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PMab-2 MAb AGAINST RAT PODOPLANIN

CHO/hPDPN, CHO/mPDPN, and P3U1 were cultured in
RPMI 1640 medium including RPMI 2 mM L-glutamine(Nacalai Tesque, Kyoto, Japan), supplemented with 10%
heat-inactivated fetal bovine serum (FBS, Thermo Fisher
Scientific), 100 U/mL penicillin, 100 mg/mL streptomycin,
and 25 mg/mL amphotericin B (Nacalai Tesque), and rat
dermal fibroblast was cultured in Fibroblast Growth Medium
2, including FBS, basic fibroblast growth factor (1 ng/mL),and insulin (5 mg/mL; PromoCell, Heidelberg, Germany) at
37C in a humidified atmosphere of 5% CO2 and 95% air.
Animals

Female BALB/c mice (four-weeks-old) were purchasedfrom CLEA Japan (Tokyo, Japan). Animals were housed
under pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved the animal experiments described herein.

397
Hybridoma production

BALB/c mice were immunized by intraperitoneal (i.p.) injection of 100 mg of synthetic peptides (rpp3851: GDDMVNPGLEDRIEC), conjugated with KLH together with Imject
Alum (Thermo Fisher Scientific). After several additional
immunizations, a booster injection 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). The culture supernatants were screened using enzyme-linked immunosorbent assay (ELISA) for binding to rpp3851 peptides.
ELISA

Synthetic peptides were immobilized on Nunc Maxisorp 96well immunoplates (Thermo Fisher Scientific) at 1 mg/mL for

FIG. 1. Specific binding of anti-PDPN MAbs. (A) Western blot analysis by PMab-2, NZ-1, and PMab-1. AC-15 (anti-bactin) and RcMAb-1 (anti-IDH1) were used as internal control. Total cell lysate were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 0.1 mg/mL of
MAbs (PMab-2, NZ-1, PMab-1) or 1 mg/mL of MAbs (AC-15 and RcMAb-1), and then with peroxidase-conjugatedsecondary antibodies; the membrane was detected using a Sayaca-Imager. (B) Flow cytometry. Cells were harvested bybrief exposure to 0.25% Trypsin/1 mM EDTA. After washing with PBS, cells were treated with PMab-2, NZ-1, and PMab1, followed by treatment with secondary antibodies. Fluorescence data were collected using a Cell Analyzer EC800.

398

30 min. After blocking with SuperBlock T20 (PBS) Blocking
Buffer, the plates were incubated with culture supernatantfollowed by 1:1000 diluted peroxidase-conjugated anti-mouse
IgG (Dako, Glostrup, Denmark). The enzymatic reaction was
conducted with a 1-Step Ultra TMB-ELISA (Thermo Fisher
Scientific). The optical density was measured at 655 nm using
an iMark microplate reader (Bio-Rad Laboratories, Philadelphia, PA). These reactions were performed with a volume of
50 mL at 37C.

OKI ET AL.
Flow cytometry

Cells were harvested by brief exposure to 0.25% Trypsin/
1 mM EDTA (Nacalai Tesque). After washing with PBS, thecells were treated with primary MAbs (1 mg/mL) for 30 min at
4C, followed by treatment with Oregon green-conjugatedanti-mouse IgG or anti-rat IgG (Thermo Fisher Scientific).
Fluorescence data were collected using a Cell Analyzer
EC800 (Sony Corp., Tokyo, Japan).

Western blot analyses

Cell lysates (10 mg) were boiled in SDS sample buffer(Nacalai Tesque). The proteins were electrophoresed on 5–
20% polyacrylamide gels (Wako Pure Chemical Industries,
Osaka, Japan) and were transferred onto a PVDF membrane(EMD Millipore, Billerica, MA). After blocking with 4%
skim milk (Nacalai Tesque), the membrane was incubatedwith primary antibodies,43 and then with peroxidaseconjugated secondary antibodies (Dako; 1:1000 diluted), anddeveloped with Pierce Western Blotting Substrate Plus
(Thermo Fisher Scientific) or ImmunoStar LD Chemiluminescence Reagent (Wako Pure Chemical Industries) using a
Sayaca-Imager (DRC Co., Tokyo, Japan).

FIG. 2. Endogenous rPDPN is detected by PMab-2. (A)Western blot analysis. AC-15 (anti-b-actin) and RcMAb-1(anti-IDH1) were used as internal control. Total cell lysate were
electrophoresed on 5–20% polyacrylamide gels and transferred
onto a PVDF membrane. After blocking, the membrane wasincubated with 1 mg/mL of MAbs, and then with peroxidaseconjugated secondary antibodies; the membrane was detectedusing a Sayaca-Imager. (B) Flow cytometry. Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA. Afterwashing with PBS, the cells were treated with PMab-2, followed by treatment with secondary antibodies. Fluorescencedata were collected using a Cell Analyzer EC800.

FIG. 3. Determination of binding affinity using flow cytometry. CHO = rPDPN, CHO = hPDPN, and CHO =mPDPN (2 · 10s cells) were re-suspended at 100 mL of serially diluted PMab-2, NZ-1, and PMab-1 (0.02–100 mg/mL,
respectively. Fluorescence data were collected using a Cell
Analyzer EC800. KD was obtained by fitting binding isotherms using built-in one-site binding models in Prism
software.

PMab-2 MAb AGAINST RAT PODOPLANIN

399

Determination of binding affinity by flow cytometry

Results

Binding affinity was determined as described previously.(44) Briefly, cells (2 · 105 cells) were resuspended at
100 mL of serially diluted antibodies (0.02–100 mg/mL), followed by secondary antibodies (Thermo Fisher Scientific).
Fluorescence data were collected using a Cell Analyzer
EC800. The dissociation constants (KD) were obtained byfitting the binding isotherms using the built-in one-sitebinding models in GraphPad PRISM 6 (GraphPad Software,
La Jolla, CA).

Production of monoclonal antibody against rPDPN

Immunohistochemical analyses

Four-mm-thick histologic sections were deparaffinized inxylene and rehydrated. Sections were incubated with PMab-2
for 1 h at room temperature followed by treatment withEnvision+ kit (Dako). Color was developed using 3,3diaminobenzidine tetrahydrochloride (DAB, Dako) for
5 min, and then the sections were counterstained with hematoxylin (Wako Pure Chemical Industries).

We first immunized mice with a synthetic peptide of rat
PDPN; then ELISA screening was performed. Among 23
ELISA-positive wells, 11 wells reacted with CHO/rPDPN
in flow cytometry. Furthermore, three wells recognized aspecific band in Western blot analysis. After limiting dilution, clone PMab-2 (mouse IgG1, kappa) was established.
PMab-2 detected about 40 kDa band of rPDPN-transfected
CHO cells (CHO/rPDPN), not with hPDPN-transfected
CHO cells (CHO/hPDPN), mPDPN-transfected CHO cells(CHO/mPDPN), and CHO cells in Western blot analysis(Fig. 1A). In contrast, NZ-1 and PMab-1 detected about
40 kDa bands of CHO/hPDPN and CHO/mPDPN, respectively, indicating that three MAbs are species-specific.
PMab-2 reacted with CHO/rPDPN, not with CHO/hPDPN,
CHO/mPDPN, and CHO cells in flow cytometry (Fig. 1B).
NZ-1 and PMab-1 specifically reacted with CHO/hPDPN
and CHO/mPDPN, respectively. Taken together, PMab-2 isa specific MAb against rPDPN, and is very useful in

FIG. 4. Immunohistochemical analysis by PMab-2. Sections of rat lung, colon, and kidney were incubated with 0.5 mg/mL
of PMab-2 or control PBS, followed by Envision+ kit, and color was developed using DAB and counterstained withhematoxylin. Arrows, lymphatic vessels; arrowheads, glomerulus. Scale bar, 100 mm.

400Western blot and flow cytometric analyses. We next investigated whether PMab-2 can detect endogenous rPDPN.
Because PDPN has been reported to be expressed in mouseor human fibroblast cells, we checked the PMab-2 reactivity
against rat dermal fibroblast. As shown in Figure 2A, PMab2 detected not only exogenous rPDPN of CHO/rPDPN but
also an endogenous one, which is expressed in rat dermal
fibroblast. PMab-2 also reacted with rat fibroblast in flow
cytometry (Fig. 2B). These results indicate that PMab-2 isuseful for detecting endogenous rPDPN. We further performed a kinetic analysis of the interaction of PMab-2 with
CHO/rPDPN using flow cytometry. As shown in Figure 3,
KD of PMab-2 was determined to be 5.9 · 10-10 M using
CHO/rPDPN. In contrast, KD of NZ-1 was determined to be
2.9 · 10-9 M using CHO/hPDPN and KD of PMab-1 wasdetermined to be 3.2 · 10-9 M using CHO/mPDPN, indi-

OKI ET AL.

cating that the binding affinity of PMab-2 is much higher
than that of NZ-1 and PMab-1.
Immunohistochemical analysis using PMab-2

We next investigated whether PMab-2 can detect rPDPN of
normal tissues in immunohistochemistry. PMab-2 reacted with
type I alveolar cells of lung, lymphatic endothelial cells of colon, podocytes, and Bowman’s capsule epithelial cells of kidney(Fig. 4). Because PMab-2 showed high affinity against rPDPN,
we next checked the dose dependency of PMab-2 in immunohistochemistry using three different tissues. As shown in Figure
5, PMab-2 detected rPDPN of lung type I alveolar cells at
0.001 mg/mL, rPDPN of colon lymphatic endothelial cells at
0.005 mg/mL, and rPDPN of kidney podocytes at 0.01 mg/mL,
indicating that PMab-2 is very sensitive against rPDPN.

FIG. 5. Immunohistochemical analysis by PMab-2 in a dose-dependent manner. Sections of rat lung, colon, and kidney
were incubated with 0.001-0.1 mg/mL of PMab-2, followed by Envision+ kit, and color was developed using DAB andcounterstained with hematoxylin. Arrows, lymphatic vessels; arrowheads, glomerulus. Scale bar, 100 mm.

PMab-2 MAb AGAINST RAT PODOPLANIN

401

Discussion

References

In this study, we immunized mice with a synthetic peptide(rpp3851), which is included in platelet aggregationstimulating (PLAG) domain of rPDPN. We previously produced an anti-hPDPN MAb (NZ-1)(17) and an anti-mPDPN
MAb (PMab-1)(39) by immunizing rats with PLAG domain of
hPDPN and mPDPN, respectively. Because these MAbs are
not only very sensitive and specific but also very useful foralmost all experiments including flow cytometry, Western
blot, immunoprecipitation, immunocytochemistry, and immunohistochemistry, MAbs against PLAG domain of rPDPN
was thought to be advantageous. As expected, establishedPMab-2 is a specific MAb against rPDPN, and is very usefulin Western blot and flow cytometric analyses. Furthermore,
PMab-2 detected not only exogenous rPDPN but also an
endogenous one of dermal fibroblast in both Western blot andflow cytometry, which leads to the advantage in immunohistochemistry. By kinetic analysis of the interaction ofPMab-2 using flow cytometry, KD of PMab-2 was determined
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lymphatic endothelial cells of colon, and podocytes of kidney in immunohistochemistry. A previous study showed that
PMab-1 detects mPDPN in immunohistochemistry; however,antigen retrieval procedure was necessary.(39) Furthermore,
NZ-1 detects hPDPN in immunohistochemistry without antigen retrieval; however, it does not react with type I alveolar
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detected rPDPN of lung type I alveolar cells at a low concentration of 1 ng/mL, indicating that PMab-2 is extraordinarily sensitive against rPDPN.
Taken together, PMab-2 is very useful for all experiments
such as flow cytometry, Western blot, and immunohistochemistry. Furthermore, the epitope of PMab-2 is PLAG
domain of rPDPN, indicating that PMab-2 is advantageousfor investigating the platelet aggregation activity of rPDPN
or rPDPN-CLEC-2 interaction.

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Acknowledgments

We thank Takuro Nakamura, Noriko Saidoh, Hazuki
Kanno, and Kanae Yoshida for their excellent technical assistance. This work was supported in part by the Platform for
Drug Discovery, Informatics, and Structural Life Science(PDIS) from Japan Agency for Medical Research and Development, AMED (Y.K.); by the Basic Science and Platform Technology Program for Innovative Biological
Medicine 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.); and by JSPS KAKENHI Grant Number 26440019(M.K.K.) and Grant Number 25462242 (Y.K.).
Author Disclosure Statement

The authors have no financial interests to disclose.

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Address correspondence to:
Yukinari Kato
Department of Regional Innovation
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku, Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: July 2, 2015
Accepted: September 15, 2015