Biochemistry and Biophysics Reports 18 (2019) 100633

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Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

Establishment of a monoclonal antibody PMab-225 against alpaca
podoplanin for immunohistochemical analyses

T

Yukinari Katoa,b,∗, Yoshikazu Furusawaa,b, Shinji Yamadab, Shunsuke Itaib, Junko Takeib,
Masato Sanob, Mika K. Kanekobab

New Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapanA R T I C LE I N FO

A B S T R A C T

Keywords:
Alpaca podoplanin
PDPN
PMab-225

Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against
human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies.
However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for
producing anti-aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1
cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using ﬂow cytometry. Oneof the mAbs, PMab-225 (IgG2b, kappa), speciﬁcally detected CHO/aPDPN cells via ﬂow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells,colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These ﬁndings demonstratethat PMab-225 antibody is useful to investigate the function of aPDPN via diﬀerent techniques.

1. Introduction
In many studies, alpaca (lama pacos) has been used for production of
antigen-speciﬁc single domain antibodies (nanobodies) [1–3]. In contrast, membrane proteins of alpaca have not been investigated due tothe lack of speciﬁc antibodies. The type I transmembrane glycoprotein,podoplanin (PDPN)/T1alpha/Aggrus, is expressed in normal tissues,
including type I lung alveolar cells, renal podocytes, and lymphatic
endothelial cells [4–6]. The interaction between PDPN on lymphatic
endothelial cells and C-type lectin-like receptor-2 (CLEC-2) on platelets
facilitates embryonic blood/lymphatic vessel separation [4,6–13]. The
expression of human PDPN (hPDPN) has been reported in several malignant tumors, including malignant brain tumors [14–17], malignant
mesotheliomas [18,19], oral squamous cell carcinomas [20], esophageal cancers [21], lung cancers [22], osteosarcomas [23–25], chondrosarcomas [24], and testicular tumors [26]. The expression of hPDPN
is associated with malignant progression and cancer metastasis[9,14,27].
We have developed monoclonal antibodies (mAbs) against human[28], mouse [28], rat [29], rabbit [30], dog [31], cat [32], bovine [33],pig [34], and horse [35] PDPNs. However, mAbs against alpaca PDPN
(aPDPN), useful for immunohistochemical analysis, remain to be developed. Sensitive and speciﬁc mAbs against aPDPN are necessary toinvestigate the expression and function of aPDPN. In the present study,
we immunized mice with CHO/aPDPN cells and established hybridomas to produce mAbs against aPDPN.
2. Materials and methods
2.1. Cell lines
CHO-K1 and P3X63Ag8U.1 (P3U1) cells were obtained from theAmerican Type Culture Collection (ATCC, Manassas, VA, USA). The
coding sequence of aPDPN bearing an N-terminal RAP16 tag (RAP16aPDPN) was inserted into a pCAG-Neo vector (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan). The RAP16 tag comprises 16
amino acids (GPGDDMVNPGLEDRIE). CHO-K1 cells were transfectedwith pCAG-Neo/RAP16-aPDPN using Lipofectamine LTX with Plus
Reagent (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA). Stabletransfectants were selected by limiting dilution and cultivating in a

Abbreviations: CBIS, Cell-Based Immunization and Screening; CHO, Chinese hamster ovary; CLEC-2, C-type lectin-like receptor-2; DAB, 3,3′-diaminobenzidine
tetrahydrochloride; aPDPN, alpaca podoplanin; hPDPN, human podoplanin; mAb, monoclonal antibody; PBS, phosphate-buﬀered saline; PDPN, podoplanin; PVDF,
polyvinylidene diﬂuoride; SDS, sodium dodecyl sulfate∗
Corresponding author. New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
E-mail address: yukinarikato@med.tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2019.100633
Received 18 December 2018; Received in revised form 2 March 2019; Accepted 27 March 20192405-5808/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license(http://creativecommons.org/licenses/BY/4.0/).

Biochemistry and Biophysics Reports 18 (2019) 100633

Y. Kato, et al.

Fig. 1. Schematic illustration of the Cell-Based Immunization and Screening (CBIS) method. Stable transfectants expressing the protein of interest are used as
immunogens with no puriﬁcation procedure. The selection of hybridomas secreting speciﬁc mAbs is performed by ﬂow cytometry using parental and transfectant
cells.

medium containing 0.5 mg/mL of G418 (Nacalai Tesque, Inc., Kyoto,
Japan).
P3U1, CHO-K1, and CHO/aPDPN cells were cultured in Roswell
Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc.).
All the media were supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientiﬁc Inc.), 100 units/mL of penicillin,
100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai
Tesque, Inc.). Cells were grown at 37 °C in a humidiﬁed environment
with an atmosphere of 5% CO2 and 95% air.

2.3. Flow cytometry

2.2. Hybridoma production

2.4. Determination of binding aﬃnity using ﬂow cytometry

Female BALB/c mice (6 weeks old) were purchased from CLEA
Japan (Tokyo, Japan). Animals were housed under speciﬁc pathogenfree conditions. The Animal Care and Use Committee of Tohoku
University approved all the animal experiments. Two BALB/c mice
were immunized with CHO/aPDPN cells (1 × 108) intraperitoneally(i.p.) administered together with Imject Alum (Thermo Fisher ScientiﬁcInc.). The procedure included three additional immunizations, followed
by a ﬁnal booster injection administered i.p. two days prior to theharvest of spleen cells, amounting to a total of ﬁve immunizations.
These spleen cells were subsequently fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN, USA), and the hybridomas were grown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher
Scientiﬁc Inc.). The cultured supernatants were screened using ﬂow
cytometry.

CHO/aPDPN was suspended in 100 μL of serially diluted PMab-225,
followed by the addition of Alexa Fluor 488-conjugated anti-mouse IgG(1:200; Cell Signaling Technology, Inc.). Fluorescence data were collected using EC800 Cell Analyzer (Sony Corp.). The dissociation constant (KD) was obtained by ﬁtting the binding isotherms to built-in onesite binding models in GraphPad PRISM 6 (GraphPad Software, Inc., La
Jolla, CA, USA).

The cells were harvested following brief exposure to 0.25% trypsin/
1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.),
washed with 0.1% bovine serum albumin (BSA)/phosphate-buﬀeredsaline (PBS), and treated with primary mAbs for 30 min at 4 °C.
Thereafter, the cells were treated with Alexa Fluor 488-conjugated antimouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA,
USA). Fluorescence data were collected using a SA3800 Cell Analyzer(Sony Corp., Tokyo, Japan).

2.5. Western blotting
Cell lysates (10 μg) were boiled in sodium dodecyl sulfate sample
buﬀer (Nacalai Tesque, Inc.). The proteins were subjected to electrophoresis on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure
Chemical Corporation) and subsequently transferred onto a polyvinylidene diﬂuoride (PVDF) membrane (Merck KGaA, Darmstadt,
Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.),2Biochemistry and Biophysics Reports 18 (2019) 100633

Y. Kato, et al.

Fig. 2. Detection of aPDPN via ﬂow cytometry using PMab-225. CHO/aPDPN CHO-K1 cells were treated with 10 μg/mL of PMab-225 (red line) or 1 μg/mL ofanti-RAP16 tag (PMab-2; blue line) or 0.1% BSA in PBS (gray) for 30 min, followed by incubation with secondary antibodies.

(FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager(DRC Co. Ltd., Tokyo, Japan).
2.6. Immunohistochemical analyses
Normal alpaca tissues were collected after autopsy at Hokkaido
University, ﬁxed in 10% neutral-buﬀered formalin [36], and routinely
processed to make paraﬃn-embedded tissue sections. Histologicalsections of 4 μm thickness were directly autoclaved in citrate buﬀer (pH
6.0; Nichirei Biosciences, Inc., Tokyo, Japan) or EnVision FLEX Target
Retrieval Solution High pH (Agilent Technologies Inc.) for 20 min.
These tissue sections were blocked using SuperBlock T20 (PBS)
Blocking Buﬀer (Thermo Fisher Scientiﬁc Inc.), incubated with PMab225 (1 μg/mL or 5 μg/mL) for 1 h at room temperature, and treatedusing an Envision + Kit (Agilent Technologies Inc.) for 30 min. Color
was developed using 3,3′-diaminobenzidine tetrahydrochloride (Agilent Technologies Inc.) for 2 min, and counterstaining was performed
using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).
3. Results
Fig. 3. Detection of aPDPN via Western blotting. Cell lysates of CHO-K1 andCHO/aPDPN (10 μg) were electrophoresed and transferred onto PVDF membranes. The membranes were incubated with l μg/mL of PMab-225, anti-RAP16
tag (PMab-2), or anti-β-actin and subsequently with peroxidase-conjugated antimouse IgG.

In this study, two mice were immunized with CHO/aPDPN cells(Fig. 1). Developed hybridomas were seeded into 96-well plates andcultivated for 8 days (ﬁrst mouse) or 9 days (second mouse). Wells
positive for CHO/aPDPN and negative for CHO-K1 were selected using
ﬂow cytometry. Screening identiﬁed strong signals against CHO/
aPDPN cells and weak or no signals against CHO-K1 cells in 83 of 960
wells (8.6%). Of these 83 wells, two hybridomas were developed. Oneof these two clones, PMab-225 (IgG2b, kappa), was selected for immunohistochemistry against alpaca tissues.
PMab-225 recognized CHO/aPDPN but showed no reaction with
CHO-K1, as assessed using ﬂow cytometry (Fig. 2). Additionally, aeach membrane was incubated with primary mouse mAbs, such as 1 μg/
mL of PMab-225, anti-RAP16 tag (PMab-2), or anti-β-actin (AC-15;
Sigma-Aldrich Corp., St. Louis, MO, USA), and subsequently with peroxidase-conjugated anti-mouse IgG (1:1000; Agilent Technologies,
Santa Clara, CA, USA). Bands were visualized using ImmunoStar LD

3

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Fig. 4. Immunohistochemical analyses against alpaca lung. Tissue sections of alpaca lung were directly autoclaved in citrate buﬀer and incubated with 1 μg/mL
of PMab-225 (A, D) or with blocking buﬀer (B, E). Type I alveolar cells were stained. (C, F) Hematoxylin and eosin staining. Scale bar = 100 μm.

kinetic analysis performed using ﬂow cytometry assessed the interaction of PMab-225 with CHO/aPDPN. KD of PMab-225 for CHO/aPDPN
cells was determined to be 2.4 × 10−9, indicating high aﬃnity for
CHO/aPDPN cells.
Western blotting performed using PMab-225 (Fig. 3) demonstrated
that PMab-225 detects aPDPN in CHO/aPDPN cells. PMab-2, an antiRAP16 tag mAb, also detected aPDPN bands in CHO/aPDPN cells.
Several bands were obtained that might represent highly glycosylated
forms.
The immunohistochemical analyses using antigen retrieval withcitrate buﬀer (pH 6.0) revealed that PMab-225 strongly stained type I
alveolar cells in the alpaca lung (Fig. 4) and lymphatic endothelial cells
in alpaca colon tissues (Fig. 5). Podocytes and Bowman's capsule ofalpaca kidney were stained using antigen retrieval with EnVision FLEX
Target Retrieval Solution High pH (Fig. 6). These results indicate that
PMab-225 will be useful to elucidate the pathophysiological functions

of aPDPN in alpaca tissues in the future.
4. Discussion
In our previous studies, we established a cancer-speciﬁc monoclonal
antibody (CasMab) technology to produce CasMabs, such as LpMab-2
and LpMab-23 against hPDPN, in several studies [17,37]. Those CasMabs against hPDPN can detect only hPDPN-expressing cancer cells,
not normal cells, including lymphatic endothelial cells and pulmonary
type I alveolar cells. Although LpMab-2 might bind to both a peptide
and glycans of hPDPN [17], LpMab-23 could detect the conformationalchange of hPDPN peptides, which might be induced by cancer-speciﬁc
glycans [38]. Both LpMab-2 and LpMab-23 possess high antitumor
activities by those antibody-dependent cellular cytotoxicities (ADCC)[38,39]. Furthermore, LpMab-23-recognizing cancer-type podoplanin
could be a novel predictor for a poor prognosis of early stage tongue
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Y. Kato, et al.

Fig. 5. Immunohistochemical analyses against alpaca colon. Tissue sections of alpaca colon were directly autoclaved in citrate buﬀer and incubated with 1 μg/
mL of PMab-225 (A, D) or with blocking buﬀer (B, E). Lymphatic endothelial cells were stained. (C, F) Hematoxylin and eosin staining. Scale bar = 100 μm.

employed the CBIS method in this study to develop sensitive and speciﬁc mAbs against aPDPN for the immunohistochemical analysis ofparaﬃn-embedded tissue sections (Fig. 1). Finally, PMab-225, which isvery useful for ﬂow cytometry (Fig. 2), Western blot (Fig. 3), and immunohistochemical analyses (Figs. 4–6), was developed. Interestingly,
PMab-225 cross-reacted with human, bovine, tiger, bear, goat, sheep,
and whale PDPNs, which were overexpressed in CHO-K1 cells (data notshown), although the percentage of homology of aPDPN with hPDPN isonly 66%. In contrast, PMab-225 did not react with mouse, rat, rabbit,
dog, cat, pig, Tasmanian devil, and horse PDPNs (data not shown). In
future study, we should determine the critical epitope of PMab-225;
then, we might uncover the mechanism of cross-reactivity against many
species. In immunohistochemical analysis, PMab-225 stained lymphatic
endothelial cells (Fig. 5) and pulmonary type I alveolar cells using
antigen retrieval with citrate buﬀer (Fig. 4). However, PMab-225 did
not stain alpaca kidney in this condition (data not shown). In contrast,

cancer [40]. Recently, we also utilized a Cell-Based Immunization andScreening (CBIS) method to establish mAbs against various membraneproteins, such as CD133 [41], CD44 [42], PD-L1 [43], pig PDPN [34],horse PDPN [44], and cat PDPN [32]. Importantly, those mAbs are very
useful for ﬂow cytometry, Western blot, and immunohistochemistry. In
contrast, we could not develop useful mAbs by immunizing synthetic
peptides (data not shown). Using selecting one method or the combination of those methods such as CasMab technology and CIBS method,
we could produce sensitive and speciﬁc mAbs against membrane proteins, which are very useful for not only ﬂow cytometry, but alsoWestern blot and immunohistochemistry when we could not develop
mAbs by immunizing synthetic peptides or recombinant proteins.
Indeed, we ﬁrst tried to produce anti-aPDPN mAbs by immunizing
synthetic peptides, which are corresponding to PLAG domains ofaPDPN; however, we could not obtain any mAbs, which are applicablefor Western blot or immunohistochemistry (data not shown). Then, we
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Y. Kato, et al.

Fig. 6. Immunohistochemical analyses against alpaca kidney. Tissue sections of alpaca kidney were directly autoclaved in EnVision FLEX Target Retrieval
Solution High pH and incubated with 5 μg/mL of PMab-225 (A, D) or with blocking buﬀer (B, E). Podocytes and Bowman's capsule were stained (C, F) Hematoxylin
and eosin staining. Scale bar = 100 μm.

Conﬂicts of interest

alpaca kidney was stained using antigen retrieval with EnVision FLEX
Target Retrieval Solution High pH (Fig. 6). In the future study, we
should clarify the molecular diﬀerence of aPDPNs, including posttranslational modiﬁcations in several tissues.
In conclusion, we have established a mAb against aPDPN, PMab225, which is suitable for use in ﬂow cytometry, Western blotting, andimmunohistochemical analyses. PMab-225 should prove useful to elucidate the pathophysiological functions of aPDPN in future studies. In
contrast, sensitive and speciﬁc mAbs against membrane proteins foralpaca have not been established; therefore, we should develop many
mAbs against alpaca membrane proteins, such as CD31 or LYVE-1 forinvestigation of vascular endothelial cells or lymphatic endothelial
cells.

The authors declare no conﬂicts of interest involving this article.
Funding
This research was supported in part by AMED under Grant
Numbers: JP18am0101078 (Y.K.), JP18am0301010 (Y.K.), andJP18ae0101028 (Y.K.), and by JSPS KAKENHI Grant Number
17K07299(M.K.K.) and Grant Number 16K10748(Y.K.).
Acknowledgments
We thank Takuro Nakamura, Miyuki Yanaka, Kayo Hisamatsu, Saori
Handa, and Yoshimi Nakamura. for their excellent technical assistance.
6

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Y. Kato, et al.

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