MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 42, Number 6, 2023
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2023.0020
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PMab-301: An Anti-Giraffe Podoplanin Monoclonal
Antibody for Immunohistochemistry

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Tsunenori Ouchida,1,* Tomohiro Tanaka,1,* Hiroyuki Suzuki,1 Kazuyuki Uchida,2 Takayuki Nakagawa,3Guanjie Li,1 Takuro Nakamura,1 Miyuki Yanaka,1 Saori Handa,1 Mika K. Kaneko,1 and Yukinari Kato1

Immunohistochemistry staining is an essential method in pathological diagnoses. Podoplanin (PDPN) is a specific maker of alveolar epithelium, lymphatic vessels, and glomeruli. In this study, we established a novel antigiraffe PDPN (girPDPN) mAb, PMab-301, using the Cell-Based Immunization and Screening (CBIS) method.
PMab-301 (mouse IgG1, kappa) detected girPDPN in various applications, such as flow cytometry, western blot,
and immunohistochemistry. PMab-301 specifically stained type-I alveolar cells using formalin-fixed paraffinembedded giraffe lung tissues. Our findings suggest the potential usefulness of PMab-301 for the pathophysiological analyses of giraffe tissues.
Keywords: giraffe, podoplanin, PDPN, PMab-301

IntroductionP

1

odoplanin (PDPN) is a mucin-type glycoprotein,which plays an essential role in the development of thelymphatic system.2 The interaction of PDPN and C-type
lectin-like receptor-2 (CLEC-2), an endogenous receptor of
PDPN,3,4 facilitates the separation of embryonic blood andlymphatic vessels.5 Human PDPN is expressed in many
malignant tumors and is associated with malignant progression and cancer metastasis via platelet aggregation.6–9 PDPN
is expressed in many cell types, including podocytes,1 pulmonary type I alveolar cells,10 and lymphatic endothelial
cells.11 In contrast, PDPN is not expressed in renal tubule
cells, pulmonary type-II alveolar cells, and vascular endothelial cells.1 Therefore, PDPN is a useful maker to distinguish these cells.
We have developed anti-PDPN mAbs for cat,12 tiger,13horse,14 pig,15 bovine,16 goat,17 sheep,18 alpaca,19 Tasmaniandevil,20 bear,21 whale,22 California sea lion,23 golden hamster,24 and ferret25 using the Cell-Based Immunization andScreening (CBIS) method.26–30 The CBIS method is aneffective method to develop mAbs for various applications,such as flow cytometry, western blot, and immunohisto-

chemistry. Here, we established a novel anti-giraffe PDPN
(girPDPN) mAb using the CBIS method.
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). girPDPN coding sequences (cds)were obtained from the WGS database (SJXV01006036.1)
by BLAST search using bovine PDPN cds as a query. The
synthesized DNA of girPDPN (Eurofins Genomics KK), in
which the original signal sequence (1-MWKVPVLFFILGSASFWVLAGA-22) was deleted, was subsequently subcloned
into a pCAGzeo_ssPA16 or ssMAP16 vector (IL2-signal
sequence and PA16 tag or MAP16 tag added to N-terminus).
The amino acid sequence of the tag system was as follows:
PA16 tag,31 16 amino acids (GLEGGVAMPGAEDDVV);
MAP16 tag,32 16 amino acids (PGTGDGMVPPGIEDKI).
The PA16 tag and the MAP16 tag can be detected by ananti-human PDPN mAb (clone NZ-1) and an anti-mouse
PDPN mAb (clone PMab-1), respectively.

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
3
Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
This paper was previously published in preprint.org (doi: 10.20944/preprints202310.0453.v1).
*Both these authors contributed equally to this work.
2

209

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The girPDPN plasmid was transfected into CHO-K1 cells,
using a Neon transfection system (Thermo Fisher Scientific,
Inc., Waltham, MA). Stable transfectants were establishedthrough cell sorting using a cell sorter (SH800; Sony Corporation, Tokyo, Japan), after which cultivation in a medium,
containing 0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA)
was conducted.
CHO-K1, girPDPN-overexpressed CHO-K1 (CHO/
MAP16-girPDPN and CHO/PA16-girPDPN), and P3U1 cells
were cultured in a Roswell Park Memorial Institute (RPMI)1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), with 10%
heat-inactivated fetal bovine serum (FBS; Thermo Fisher
Scientific, Inc.), 100 units/mL of penicillin, 100 lg/mL of
streptomycin, and 0.25 lg/mL of amphotericin B (Nacalai
Tesque, Inc.). All cells were grown in a humidified incubator
at 37C, in an atmosphere of 5% CO2 and 95% air.

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Hybridomas

Two 5-week-old BALB/c mice were purchased from
CLEA Japan (Tokyo, Japan). The animals were housed under
specific pathogen-free conditions. All animal experiments
were performed according to the relevant guidelines andregulations to minimize animal suffering and distress in thelaboratory. The Animal Care and Use Committee of Tohoku
University (Permit number: 2022MdA-001) approved animal
experiments. The mice were monitored daily for healthduring the complete 4-week duration of the experiment.
A reduction of more than 25% of the total body weight was
defined as a humane endpoint. During the sacrifice, the mice
were euthanized through cervical dislocation, after which
deaths were verified through respiratory and cardiac arrest.
To develop mAbs against girPDPN, we intraperitoneallyimmunized two mice with CHO/MAP16-girPDPN cells(1 · 108 cells/mouse) plus Imject Alum (Thermo Fisher Scientific, Inc.). The procedure included three additional injections every week (1 · 108 cells/mouse), which was followed
by a final booster intraperitoneal injection (1 · 108 cells/mouse), 2 days before harvesting splenocytes. The harvested
splenocytes were subsequently fused with P3U1 cells, using
PEG1500 (Roche Diagnostics, Indianapolis, IN). For the
hybridoma selection, cells were cultured in the RPMI-1640
medium with 10% FBS, 100 units/mL of penicillin,
100 lg/mL of streptomycin, 0.25 lg/mL of amphotericin B,
5 lg/mL of plasmocin, 5% Briclone (NICB, Dublin, Ireland),and hypoxanthine, aminopterin, and thymidine (HAT;
Thermo Fisher Scientific, Inc.). The supernatants were subsequently screened using flow cytometry using CHO/PA16girPDPN and CHO-K1.
Purification of mAbs

The cultured supernatants of PMab-301-producing hybridomas were filtrated with Steritop (0.22 lm; Merck KGaA,
Darmstadt, Germany). The filtered supernatants were subsequently applied to 1 mL of Ab-Capcher ExTra (ProteNova,
Inc., Kagawa, Japan). After washing with phosphate-buffer
saline (PBS), bound antibodies were eluted with an IgGelution buffer (Thermo Fisher Scientific, Inc.), followed by
immediate neutralization of eluates, using 1 M Tris-HCl(pH 8.0). Finally, the eluates were concentrated, after which
PBS was replaced with the elution buffer using Amicon Ultra(Merck KGaA).

OUCHIDA ET AL.
Flow cytometric analyses

CHO-K1 and CHO/PA16-girPDPN cells were harvested
after a brief exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). The
cells were subsequently washed with 0.1% bovine serum
albumin (BSA) in PBS and treated with 1 lg/mL NZ-1 orPMab-301 for 30 minutes at 4C. The cells were treated with
2 lg/mL of Alexa Fluor 488-conjugated anti-rat IgG (Cell
Signaling Technology, Inc., Danvers, MA) for NZ-1 or Alexa
Fluor 488-conjugated anti-mouse IgG (Cell Signaling Technology, Inc.) for PMab-301. The fluorescence data were collected using the SA3800 Cell Analyzer (Sony Corporation).
To determine the dissociation constant (KD), PMab-301
was serially diluted from 10 mg/mL to 0.61 ng/mL. The
geometric mean of fluorescence intensity of CHO/PA16girPDPN at each concentration was calculated by FlowJo
v10.8.1 (Becton, Dickinson & Company, Ashland, OR). The
KD was estimated by fitting saturation binding curves to thebuilt-in; one-site binding models in GraphPad PRISM 8(GraphPad Software, Inc., La Jolla, CA).
Western blotting

Cell lysates (10 lg) were boiled in sodium dodecyl sulfate(SDS) 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). After blocking with 4% skim milk
(Nacalai Tesque, Inc.) in 0.05% Tween 20-containing PBS,
membranes were incubated with 5 lg/mL of PMab-301,
1 lg/mL of an anti-PA16 tag mAb (NZ-1), or 1 lg/mL of ananti-IDH1 mAb (RcMab-1). IDH1 is an internal control.
Then, they were incubated again with horseradish peroxidaseconjugated anti-mouse immunoglobulins (for PMab-301;
diluted 1:1,000; Agilent Technologies, Inc., Santa Clara, CA)or anti-rat immunoglobulins (for NZ-1 and RcMab-1; diluted
1:10,000; Sigma-Aldrich Corporation, St. Louis, MO).
Finally, protein bands were detected using ECL Plus Western
Blotting Substrate (Thermo Fisher Scientific, Inc.) and a
Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
Immunohistochemical analyses

Giraffe lung tissue samples obtained from routine necropsies performed at the Laboratory of Veterinary Pathology,
the University of Tokyo, fixed in 10% neutral-bufferedformalin, were processed to make formalin-fixed paraffinembedded (FFPE) tissue sections. To deparaffinize, rehydrate, and retrieve antigen, the sections were autoclaved in
Deparaffinization/Antigen Retrieval Solution pH6 (low pH;
No. 415281, Nichirei Biosciences, Tokyo, Japan) 121C for
20 minutes. Then, sections were blocked using the Super
Block T20 (PBS) Blocking Buffer (Thermo Fisher Scientific,Inc.), incubated with 5 lg/mL PMab-301 for 1 hour at room
temperature, and treated with the EnVision + Kit for mouse(Agilent Technologies, Inc.) for 30 minutes at room temperature. Finally, Color was developed using 3,3¢-diaminobenzidine
tetrahydrochloride (DAB; Agilent Technologies, Inc.) for
5 minutes, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).
Hematoxylin and eosin (H&E) staining was performed
using the serial sections.

ESTABLISHMENT OF ANTI-GIRAFFE PDPN MAB
Results and Discussion
Establishment of anti-girPDPN mAbs

PMab-301 reacted girPDPN-overexpressing CHO-K1
in flow cytometry

We checked the reactivity of PMab-301 to girPDPN byflow cytometry. PMab-301 reacted to CHO/PA16-girPDPN
cells in a dose-dependent manner, but did not react with
CHO-K1 cells (Fig. 2A). To determine the KD of PMab-301
against CHO/PA16-girPDPN cells, we conducted kinetic
analysis by flow cytometry. The geometric mean of the fluorescence intensity was plotted versus the concentration ofPMab-301. The KD value of PMab-301 for girPDPN was
determined as 1.3 · 10-8 M (Fig. 2B).

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We immunized mice with girPDPN-overexpressing
CHO-K1 (CHO/MAP16-girPDPN) cells (Fig. 1A). To produce hybridomas, the splenocytes from these mice werefused with P3U1 cells using polyethylene glycol (Fig. 1B).
To select anti-girPDPN mAb-producing hybridomas, thereactivities to CHO/PA16-girPDPN cells were observed byflow cytometry (Fig. 1C). After limiting dilution, PMab-301(mouse IgG1, kappa) was finally established (Fig. 1D).

211

FIG. 1. The establishment of PMab-301 using the CBIS method. (A) MAP16-tagged girPDPN-overexpressing CHO-K1(CHO/MAP16-girPDPN) cells were immunized into two BALB/c mice using an intraperitoneal injection. (B) The splenocytes were fused with P3U1 cells. (C) The culture supernatants were screened through flow cytometry to select antigirPDPN mAb-producing hybridomas. (D) PMab-301 was established through limiting dilution and some additional
screenings. CBIS, Cell-Based Immunization and Screening; CHO, Chinese hamster ovary; girPDPN, giraffe podoplanin.

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FIG. 2. Flow cytometry of girPDPN-overexpressing cells using PMab-301. (A) CHO/PA16-girPDPN and CHO-K1 cells
were treated with 0.01–10 mg/mL of PMab-301 (red line) or blocking buffer (negative control, black line), followed bytreatment with AlexaFluor488-conjugated anti-mouse IgG. (B) The binding affinity of PMab-301 was determined against
CHO/PA16-girPDPN cells by flow cytometry. The dots show the geometric mean of fluorescence intensity of CHO/PA16girPDPN at each concentration.

212

ESTABLISHMENT OF ANTI-GIRAFFE PDPN MAB

213

girPDPN was detected by western blot by PMab-301

We examined whether PMab-301 is applicable for westernblot. Due to glycosylation, PDPN has been detected as about
a 48 kDa-band.18,19,33 As demonstrated in Figure 3, PMab301 and NZ-1 detected the 48-kDa band of girPDPN in
lysates from CHO/PA16-girPDPN cells, whereas this band
was absent in lysates of CHO-K1 cells. These data indicatethat PMab-301 specifically detects girPDPN in western blot.

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PMab-301 recognized girPDPN
in immunohistochemistry

Finally, we investigated whether PMab-301 can be used
for immunohistochemical analyses using FFPE giraffe lungtissue sections because PDPN is known to be expressed on
pulmonary type I alveolar cells of various species.10
As shown in Figure 4A and 4B, PMab-301 strongly stained
alveolar epithelial cells, but did not react with vascularendothelial cells, indicating that PMab-301 is useful for
detecting girPDPN in immunohistochemistry.
We successfully established PMab-301 against girPDPN
by the CBIS method and assessed the application of PMab301. PMab-301 could recognize girPDPN in flow cytometry,western blot, and immunohistochemistry, indicating that
PMab-301 can be used in various applications to detect
girPDPN.
Several studies have reported that many giraffes diedfrom unknown causes.34–36 Immunohistochemistry is a com-

FIG. 4. Immunohistochemical staining of giraffe lungwith PMab-301. Histological sections of the giraffe lungwere autoclaved in a citrate buffer and incubated with
5 mg/mL of PMab-301 (A, B) or a blocking buffer (C, D),followed by the EnVision+Kit. (E, F) H&E staining was
performed. Scale bars = 100 mm. H&E, hematoxylin andeosin.
monly used method in pathological examination to find out
the cause of death. In this study, PMab-301 could stain
alveolar epithelium cells, but did not react with vascularendothelial cells in giraffe lung tissues (Fig. 4). These data
indicate that PMab-301 could bind to girPDPN specificallyin immunohistochemistry of giraffe tissue. Because PDPN isa maker of not only alveolar epithelium but also lymphatic
endothelial cells in various animals,16–19 PMab-301 will be ahelpful antibody to distinguish lymphatic endothelial cells
from vascular endothelial cells in giraffe tissues.
Authors’ Contributions

T.O., T.T., H.S., G.L., T. Nakamura, M.Y., and S.H. performed the experiments. K.U., T. Nakagawa, M.K.K., andY.K. designed the experiments. H.S. and M.K.K. analyzed thedata. T.O. and Y.K. wrote the manuscript. All authors haveread and agreed to the published version of the manuscript.
Author Disclosure Statement

FIG. 3. Western blot with PMab-301. Cell lysates (10 mg)
of CHO-K1 and CHO/PA16-girPDPN cells were electrophoresed, after which proteins were transferred to PVDF
membranes. After blocking, membranes were subsequently
incubated with PMab-301, anti-PA16 tag mAb (NZ-1), or
anti-IDH1 mAb (RcMab-1). RcMab-1 was used to detect aninternal control. They were incubated with horseradish
peroxidase-conjugated anti-mouse immunoglobulins (forPMab-301) or anti-rat immunoglobulins (for NZ-1 andRcMab-1).

The authors have no conflict of interest.
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under Grant
numbers: JP23ama121008 (to Y.K.), JP23am0401013(to Y.K.), 23bm1123027h0001 (to Y.K.), and JP23ck0106730(to Y.K.), and by the Japan Society for the Promotion of

214

Science ( JSPS) Grants-in-Aid for Scientific Research(KAKENHI) grant numbers 23K19494 (to T.O.), 21K20789(to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and22K07224 (to Y.K.).

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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: November 7, 2023
Accepted: December 11, 2023