MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 4, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0007
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Development of a Monoclonal Antibody PMab-295 Against
Elephant Podoplanin

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Yuma Kudo,1,* Hiroyuki Suzuki,1,* Mika K. Kaneko,2 and Yukinari Kato1,2

Podoplanin (PDPN) is an essential marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) that can specifically recognize PDPN in immunohistochemistry areimportant to analyze the development of tissues and the pathogenesis of diseases, including cancers. We havedeveloped anti-PDPN mAbs against many animal species; however, mAbs that can recognize elephant-derived
membrane proteins and distinguish the specific cell types in immunohistochemistry are limited. In this study, anovel anti-elephant PDPN (elePDPN) mAb, PMab-295 (IgG1, kappa), was established using the peptide immunization method. PMab-295 recognized both elePDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells andendogenous elePDPN-expressed LACF-NaNaI cells by flow cytometry and western blotting. Kinetic analyses
using flow cytometry showed that the KD of PMab-295 for CHO/elePDPN was 1.5 · 10-8 M. Furthermore, PMab295 detected elePDPN-expressing cells using immunohistochemistry. These results showed the usefulness ofPMab-295 to investigate the molecular function of elePDPN and the pathogenesis of diseases.
Keywords: elephant podoplanin, monoclonal antibody, immunohistochemistry

IntroductionP

odoplanin (PDPN) is a mucin-type type I transmembrane glycoprotein and is known as an important marker
of lung type I alveolar cells,(1,2), kidney podocytes,(3) andlymphatic endothelial cells.(4,5) In humans, PDPN has beenreported to be overexpressed in cancers and plays crucial
roles in cancer cell migration, invasion, and metastasis.(6)Therefore, anti-PDPN monoclonal antibodies (mAbs) areuseful to distinguish the specific cell types and investigate the
PDPN-expressing cancer cells.
We have developed anti-PDPN mAbs for cat,(7) tiger,(8)horse,(9) pig,(10) goat,(11) alpaca,(12) Tasmanian devil,(13)bear,(14) whale,(15) sheep,(16) California sea lion,(17) golden
hamster,(18,19) and ferret(20) using the Cell-Based Immunization and Screening (CBIS) method. These mAbs are available
for flow cytometry, western blotting, and immunohistochemistry. However, an anti-elephant PDPN (elePDPN) mAb(clone PMab-265), which was established by the CBIS
method, can be applicable only for flow cytometry, but not forthe other applications.(21) In this study, we used the pep-tide immunization methods to obtain novel mAbs against
elePDPN and investigated the several applications, including
flow cytometry, western blotting, and immunohistochemistry.
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture Collection (Manassas, VA). African savanna elephantfibroblast-like cells (LACF-NaNaI) were obtained from the
RIKEN BioResource Center (Ibaraki, Japan).(21) The elePDPN
ORF with N-terminal RIEDL tag (RIEDL-elePDPN) was expressed in CHO-K1 cells, and previously named as CHO/
elePDPN.(21) P3U1, CHO-K1, and CHO/elePDPN cells were
cultured in RPMI-1640 (Nacalai Tesque, Inc., Kyoto, Japan)
supplemented with 10% heat-inactivated fetal bovine serum(FBS; Thermo Fisher Scientific, Inc.), 100 U/mL penicillin,
100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B(Nacalai Tesque, Inc.) and were incubated at 37C in a

Departments of 1Molecular Pharmacology and 2Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Japan.
*These authors contributed equally to this work.

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DEVELOPMENT OF ANTI-ELEPHANT PDPN MAB

humidified atmosphere containing 5% CO2. LACF-NaNaI
cells were cultured in minimum essential medium (Nacalai
Tesque, Inc.), supplemented with 10% heat-inactivated FBS(Thermo Fisher Scientific, Inc.), 100 U/mL penicillin,
100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B(Nacalai Tesque, Inc.), 5 lg/mL Plasmocin (InvivoGen), and
1 mM L-sodium pyruvate (Nacalai Tesque, Inc.) and wereincubated at 37C in a humidified atmosphere containing 5%
CO2.

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Hybridoma production

Female BALB/c mice (6-week 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 animal experiments. The elePDPN peptide immunization method wasused to develop mAbs against elePDPN. Briefly, two mice
were immunized, using three keyhole limpet hemocyaninconjugated elePDPN peptides (100 lg of each peptide),
including 38-EGGMVIPGVEDNMV-51, + C-terminal cyste-

195

ine. The administration was conducted through the intraperitoneal route with an Imject Alum (Thermo Fisher Scientific,Inc.). The procedure included three additional immunization procedures (100 lg of each peptide), followed by a final
booster intraperitoneal injection (100 lg of each peptide) 2
days before its spleen cells were harvested. The harvested
spleen cells were subsequently fused with P3U1 cells, using
polyethylene glycol 1500 (PEG1500; Roche Diagnostics,
Indianapolis, IN). Then, hybridomas were grown in an RPMI
medium supplemented with hypoxanthine, aminopterin, andthymidine for selection (Thermo Fisher Scientific, Inc.).
Cultured supernatants were finally screened using enzymelinked immunosorbent assay (ELISA) and flow cytometricanalysis.
Enzyme-linked immunosorbent assay

Synthesized elePDPN peptides (38-EGGMVIPGVEDN
MV-51, + C-terminal cysteine) were immobilized on Nunc
MaxiSorp 96-well immunoplates (Thermo Fisher Scientific,Inc.) at a concentration of 1 lg/mL for 30 minutes at 37C.

FIG. 1. A schematic procedure of anti-elePDPN mAb production. Mice were intraperitoneally immunized with theelePDPN peptide. The screening was then conducted by ELISA and flow cytometry. elePDPN, elephant PDPN; ELISA,
enzyme-linked immunosorbent assay; mAbs, monoclonal antibodies.

196

After washing with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (Nacalai Tesque, Inc.), wells were
blocked with 1% bovine serum albumin (BSA)-containing
PBST for 30 minutes at 37C. Culture supernatants wereadded to each well, followed by peroxidase-conjugated antimouse immunoglobulins (1:2000 diluted; Agilent Technologies, Inc., Santa Clara, CA). Afterward, enzymatic reactions
were conducted, using the ELISA POD Substrate TMB Kit(Nacalai Tesque, Inc.), followed by the measurement of the
optical density at 655 nm, using an iMark microplate reader(Bio-Rad Laboratories, Inc., Berkeley, CA).
Flow cytometry

cells were treated with PMab-295 (1 lg/mL) for 30 minutes at
4C, followed by Alexa Fluor 488-conjugated anti-mouse
IgG (1:1000; product no. 4408; Cell Signaling Technology,
Inc., Danvers, MA). Finally, fluorescence data were collectedusing SA3800 Cell Analyzer (Sony Biotechnology Corp.,
Tokyo, Japan).
Determining the binding affinity of cells

Cells were suspended in 100 lL serially-diluted PMab-295(0.006–100 lg/mL), followed by a subsequent suspension inan Alexa Fluor 488-conjugated anti-mouse IgG solution(1:200; Cell Signaling Technology, Inc.). Then, fluorescencedata were collected, using the BD FACSLyric (BD Biosciences,Franklin Lakes, NJ). By fitting binding isotherms to built-in
one-site binding models in GraphPad Prism 8 (GraphPad
Software, Inc., La Jolla, CA), the dissociation constant (KD) was
finally calculated.

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Cells (2 · 105 cells/mL) were harvested after brief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic
acid (EDTA; Nacalai Tesque, Inc.). After washing with 0.1%
BSA (Nacalai Tesque, Inc.) in PBS (Nacalai Tesque, Inc.),

KUDO ET AL.

FIG. 2. Flow cytometry using anti-elePDPN mAb, PMab-295. (A) CHO-K1 and CHO/elePDPN cells were treated with 1–
0.001 mg/mL of PMab-295, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG. The black line
represents the negative control. (B) LACF-NaNaI cells were treated with 1 mg/mL of PMab-295, followed by treatment with
Alexa Fluor 488-conjugated anti-mouse IgG. The black line represents the negative control. (C) CHO/elePDPN cells weresuspended in 100 mL serially-diluted PMab-295 (100–0.0006 mg/mL), following subsequent treatment with Alexa Fluor 488conjugated anti-mouse IgG. Fluorescence data were subsequently collected using a BD FACSLyric, after which we
calculated the dissociation constant (KD) using GraphPad PRISM 8. CHO, Chinese hamster ovary.

DEVELOPMENT OF ANTI-ELEPHANT PDPN MAB
Western blotting

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Cell lysates (10 lg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.), after which proteins
were separated on 5%–20% polyacrylamide gels (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan) and transferred to polyvinylidene difluoride membranes (Merck
KGaA, Darmstadt, Germany). After blocking with 4% skim
milk (Nacalai Tesque, Inc.) in 0.05% Tween 20-containing
PBS, membranes were incubated with 1 lg/mL of PMab-295
or an anti-isocitrate dehydrogenase 1 (IDH1) mAb (RMab3).(22–25) Then, they were incubated again with peroxidaseconjugated anti-mouse immunoglobulins (diluted 1:1000;
Agilent Technologies, Inc.). Finally, protein bands were detected using ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) and a Sayaca-Imager (DRC Co. Ltd.,Tokyo, Japan).

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295 interactions with CHO/elePDPN cells was 1.5 · 10-8 M,
suggesting that PMab-295 exhibited a moderate affinity
against elePDPN.
Western blotting

Western blotting was conducted to further assess the sensitivity of PMab-295. For this assessment, lysates of CHOK1, CHO/elePDPN, and LACF-NaNaI cells were probed. As
demonstrated in Figure 3, PMab-295 detected double bands(between 35 and 48 kDa) of elePDPN in lysates from
CHO/elePDPN cells, whereas these bands were absent inlysates from CHO-K1 cells. Furthermore, PMab-295 detected the endogenous elePDPN (about 40 kDa) in lysatesfrom LACF-NaNaI cells. These results indicated that PMab295 specifically detected both exogenous and endogenous
elePDPN by western blotting.

Immunohistochemical analysis

Immunohistochemical analyses

Cell blocks were produced using iPGell (Genostaff Co.,
Ltd., Tokyo, Japan)(13) and processed to make 4 lm thickparaffin-embedded cell sections that were directly autoclavedin a citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo,
Japan) for 20 minutes. These tissue sections were blocked
using the SuperBlock T20 (PBS) Blocking Buffer (Thermo
Fisher Scientific, Inc.), incubated with PMab-295 (5 lg/mL)
for 1 hour at the room temperature, and then treated with the
EnVision+Kit (Agilent Technologies, Inc.) for 30 minutes.
Color was developed using 3,3¢-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes,
and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

We next investigated whether PMab-295 can be used for
immunohistochemical analyses using paraffin-embedded cell
(CHO-K1, CHO/elePDPN, and LACF-NaNaI cells) sections.
PMab-295 strongly stained the CHO/elePDPN cell-embedded
section, but not CHO-K1-embedded section (Fig. 4A, B).

Results
Establishment of anti-elePDPN mAbs

To develop anti-elePDPN mAbs, we used the elePDPN
peptide immunization method. We designed the peptide in
the platelet aggregation-stimulating domain of elePDPN
since we could successfully generate anti-PDPN mAbs invarious species using this strategy.(26–37) Hybridoma screening was conducted using ELISA and flow cytometry (Fig. 1).
Mice were immunized with elePDPN synthetic peptides.
Subsequently, hybridomas were seeded into 96-well plates,
after which ELISA was used to extract positive wells for
elePDPN peptides, followed by the selection of CHO/
elePDPN-reactive and CHO-K1-nonreactive supernatants
using flow cytometry. Finally, we established seven clones,
which are applicable for flow cytometry. Among them, weselected PMab-295 (mouse IgG1, kappa) for further analysis.
Flow cytometric analyses

Flow cytometric analyses were conducted using PMab295 with CHO/elePDPN and CHO-K1 cells. PMab-295
recognized CHO/elePDPN dose dependently. In contrast, it
did not react with CHO-K1 cells (Fig. 2A). PMab-295 also
reacted with endogenous elePDPN, which is expressed in an
African savanna elephant fibroblast-like cell line, LACFNaNaI (Fig. 2B). Furthermore, kinetic analysis of PMab-295
interactions with CHO/elePDPN cells was conducted using
flow cytometry. As indicated in Figure 2C, the KD for PMab-FIG. 3. Western blotting using PMab-295. Cell lysates(10 mg) of CHO-K1 and CHO/elePDPN and LACF-NaNaI
cells were electrophoresed, after which proteins were transferred to PVDF membranes. After blocking, membranes
were subsequently incubated with 1 mg/mL of PMab-295 oranti-IDH1 (RMab-3), following incubation with peroxidaseconjugated anti-mouse immunoglobulins. IDH1, isocitrate
dehydrogenase 1; PVDF, polyvinylidene difluoride.

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KUDO ET AL.

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endothelial marker. Furthermore, PMab-295 will contributeto the analyses of the injured lung and kidney in the future.
The incidence of elephant cancer is known to be extremely
rare. However, about 3% of elephants develop cancer.(42,43)
PDPN is overexpressed in many human cancers, including
squamous cell carcinomas (head and neck,(44) lung,(45) uterine,(46) oral,(47) and esophageal(48) carcinomas), malignant
mesotheliomas,(49–55) sarcomas,(56,57) and malignant gliomas.(26,58–65) Recent study reported the reproductive tract
neoplasia in 80 adult female Asian elephant mortalities in the
United States from 1988 to 2019. Neoplasms occurred in 64/
80 (80%) of cases. Most were in the uterus (63/64; 98%) with
only a single case of ovarian neoplasia. This study also investigated the expression of pan-cytokeratin, vimentin, andestrogen receptor in the uterine carcinomas by immunohistochemistry.(66) Therefore, the analysis of elephant carcinomas by PMab-295 will help the elucidation of the mechanismof cancer development and the classification of neoplasia.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

FIG. 4. Immunohistochemical analyses. The paraffinembedded cell sections of CHO-K1 (A, B) and CHO/
elePDPN (C, D) and LACF-NaNaI (E, F) cells were incubated with 5 mg/mL of PMab-295 (A, C, E) or with blocking
buffer control (B, D, F), followed by that with the Envision+Kit. Arrows show elePDPN-expressing LACF-NaNaI
cells. Scale bar = 100 mm.
Moreover, PMab-295 weakly stained the LACF-NaNaI cellembedded section (Fig. 4C). These results indicate the usefulness of PMab-295 for detecting elePDPN-positive cells inparaffin-embedded cell samples.
Discussion

Previously, we established an anti-elePDPN mAb (clone
PMab-265), which is only applicable for flow cytometry.(21)In this study, we established a novel anti-elePDPN mAb(clone PMab-295), which is useful for flow cytometry,western blotting, and immunohistochemistry. Especially,
PMab-295 could stain paraffin-embedded cells (Fig. 4).
Therefore, PMab-295 can be used to detect and analyze thedevelopment and pathogenesis of elephant tissues. Further
studies are required to show whether PMab-295 can identify
the elephant lung type I alveolar cells, kidney podocytes, andlymphatic endothelial cells.
Recently, the pathogenesis of elephant endotheliotropic
herpesvirus-hemorrhagic disease has been reported as themost profound viral infectious disease in young Asian elephants (Elephas maximus).(38–41) These studies investigatedthe pathogenesis of the affected tissues, including heart, liver,lung, kidney, and intestine by hematoxylin and eosin staining
and immunohistochemistry. Especially a study showed theincreases in platelet endothelial cell adhesion molecules-1,
and von Willebrand factor positive cells were significantly
observed in injured blood vessels.(40) PMab-295 could contribute to the analyses of the pathogenesis as a lymphatic

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under grantnos: JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), andJP21am0101078 (to Y.K.).
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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: February 4, 2022
Accepted: July 3, 2022