MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 3, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0016

Ferret Podoplanin Is Detected by PMab-241
in Immunohistochemistry

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

Ren Nanamiya,1,* Junko Takei,1,2,* Teizo Asano,1,* Masato Sano,1 Tomohiro Tanaka,1
Hideki Hosono,1 Hiroyuki Harada,2 Yusuke Sakai,3 Takuya Mizuno,4,i Hiroyoshi Suzuki,5Mika K. Kaneko,1 and Yukinari Kato1,6,ii

Podoplanin (PDPN) plays an important role in the development of many normal tissues and is expressed invarious cancers. We have previously developed multiple monoclonal antibodies (mAbs) against PDPNs from avariety of animal species and characterized each of these PDPNs using the anti-PDPN mAbs. In this study, weevaluated whether these anti-PDPN mAbs possess cross-reactivity with ferret PDPN (ferPDPN) using flow
cytometry. Comprehensive analysis using 17 differing anti-PDPN mAbs available for immunohistochemistryuse, demonstrated that the anti-bear PDPN mAb (clone PMab-241) strongly cross-reacts with ferPDPNoverexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells. Immunohistochemistry analysis demonstrated
intense PMab-241 staining within Bowman’s capsules and glomeruli of the ferret kidney, and lymphatic
endothelial cells of the ferret lung. These results demonstrate that PMab-241 is suitable for the detection of
PDPN in ferret tissues.
Keywords: ferret podoplanin, PDPN, immunohistochemistry

IntroductionP

odoplanin (PDPN)/T1a/gp36/Aggrus is a type I
transmembrane sialoglycoprotein(1) that plays essential
roles in the development of the heart,(2) alveoli,(3) and lymphatic system.(4) Alongside its roles in normal tissue development, it is also expressed in some cancer cells, including
brain tumors,(5) bladder cancers,(6) and squamous cell lungcarcinomas.(7) PDPN has been shown to induce platelet aggregation by binding to the C-type lectin-like receptor-2(CLEC-2)(8,9) and this dynamic between PDPN/CLEC-2 axis
in platelets, is what regulates inflammation.(10) In addition,
PDPN has been reported to be associated with various proteins, including galectin 8,(11) CD44,(12) heat-shock protein
A9,(13) and CC chemokine ligand 21.(14) The expression of
PDPN is induced by tumor promoters such as Src andTPA.(15,16) Miyashita et al. reported that PDPN activates

Rho-associated coiled-coil kinase (ROCK) activity, and thisspecific activation by PDPN suppresses cell apoptosis.(17)This was demonstrated with a reduction in PDPN-positive
cells upon treatment with a ROCK inhibitor. Sikorska et al.
demonstrated the link between PDPN and the epithelialmesenchymal transition through regulation of ezrin, radixin,
and moesin expression, or matrix metalloproteinases-2
and -9.(18) This report also indicated the inhibition of the
MAPK pathway resulted in a reduced level of PDPN expression in thyroid cancer cell lines. These reports demonstrate that PDPN is involved in the control of tumor cell
migration, invasion, and metastasis.
PDPN is expressed in not only podocytes of kidneys, butalso within type I alveolar cells of the lung and lymphatic
endothelial cells of various tissues. Therefore, anti-PDPN
monoclonal antibodies (mAbs) are valuable in distinguishing between lymphatic endothelial cells from vascular1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
3
Laboratory of Veterinary Pathology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
4
Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
5
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
6
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
*Contributed equally to this study.
i
ORCID ID (https://orcid.org/0000-0002-9580-7557).
ii
ORCID ID (https://orcid.org/0000-0001-5385-8201).
2

134

FERRET PDPN IN IMMUNOHISTOCHEMISTRY

endothelial cells, or between type I alveolar cells to type II
alveolar cells of the lung.(1,19) We have previously developed various anti-PDPN mAbs for human,(20–22) mouse,(20)rat,(23) rabbit,(24) dog,(25,26) bovine,(27) cat,(28) tiger,(29)horse,(30,31) pig,(32) goat,(33) alpaca,(34) Tasmanian devil,(35) bear,(36,37) whale,(38) and sheep.(39) However, antiferret PDPN (ferPDPN) mAbs have not yet been reported.
In this study, we investigated whether those previously
reported anti-PDPN mAbs could have cross-reactivity with
ferPDPN using flow cytometry and immunohistochemical
analyses.
Materials and Methods

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

Cell lines

Chinese hamster ovary (CHO)-K1 cells were obtained
from the American Type Culture Collection (ATCC, Manassas, VA). Synthesized DNA (Eurofins Genomics KK,Tokyo, Japan) encoding ferPDPN plus an N-terminal MAP
tag (GDGMVPPGIEDK),(40) which is recognized by an antiMAP tag mAb (PMab-1), were subcloned into a pCAG-Ble
vector (Fujifilm Wako Pure Chemical Corporation, Osaka,
Japan). Plasmids were transfected into CHO-K1 using
Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants (CHO/
MAP-ferPDPN) were selected using cell sorter (SH800; Sony
Corp., Tokyo, Japan).
CHO-K1 and CHO/MAP-ferPDPN were cultured in a
Roswell Park Memorial Institute 1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan), supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific,Inc.), 100 U/mL of penicillin, 100 lg/mL of streptomycin,
and 0.25 lg/mL of amphotericin B (Nacalai Tesque, Inc.).
CHO/MAP-ferPDPN were cultivated in a medium containing 0.5 mg/mL Zeocin (InvivoGen, San Diego, CA). The
cells were cultured in an incubator at 37C with humidity and
5% CO2 and 95% air atmosphere.
Ferret tissues

Ferret (Mustela putorius furo) tissues were collected after
autopsy at Yamaguchi University, and fixed in 10% neutralbuffered formalin. Then, paraffin-embedded tissue sections(4-mm thick) were made.
Flow cytometry

CHO/MAP-ferPDPN cells were harvested after short-time
exposure to 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.), and were subsequently
washed with 0.1% bovine serum albumin in phosphatebuffered saline (PBS). Cells were treated with primary mAbs
(10 lg/mL) for 30 minutes at 4C. The primary mAbs usedwere as follows: PMab-2 for rat PDPN,(23) PMab-32 for
rabbit PDPN,(24) PMab-38 for dog PDPN,(25) PMab-44

135

for bovine PDPN,(27) PMab-48 for dog PDPN,(26) PMab-52
for cat PDPN,(28) PMab-210 for pig PDPN,(41) PMab-213 for
pig PDPN,(32) PMab-219 for horse PDPN,(31) PMab-225 foralpaca PDPN,(34) PMab-231 for tiger PDPN,(29) PMab-233
for Tasmanian devil PDPN,(35) PMab-235 for goat PDPN,(33)PMab-237 for whale PDPN,(38) PMab-241 for bear PDPN,(37)PMab-247 for bear PDPN,(36) and PMab-256 for sheep
PDPN.(39) After incubation with primary antibodies, cells
were treated with Alexa Fluor 488-conjugated anti-mouse
IgG (1:2000; Cell Signaling Technology, Inc., Danvers,
MA). An anti-MAP tag mAb (clone PMab-1; 10 lg/mL) was
also used for detecting N-terminal MAP tag,(40) followed
by Alexa Fluor 488-conjugated anti-rat (H+L) IgG (1:2000;
Cell Signaling Technology, Inc.). Fluorescence data werecollected using the SA3800 Cell Analyzer (Sony Corp.).
Immunohistochemical analyses

Histological sections (4-lm thick) of ferret tissue weredirectly autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 minutes. After blocking
histological sections with the SuperBlock T20 (PBS)
Blocking Buffer (Thermo Fisher Scientific, Inc.), sections
were incubated with PMab-241 (10 lg/mL) for 1 hour atroom temperature and treated with the EnVision + Kit formouse (Agilent Technologies, Inc., Santa Clara, CA), for 30
minutes. Color was developed with 3,3¢-diaminobenzidine
tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed using hematoxylin(Fujifilm Wako Pure Chemical Corporation). Hematoxylin
and eosin (HE; Fujifilm Wako Pure Chemical Corporation)
staining was also performed using the serial section of theferret tissues.
Results
Flow cytometry analyses using anti-PDPN mAbs
for ferPDPN

To begin, a stable transfectant cell line of ferPDPN with
N-terminal MAP tag (CHO/MAP-ferPDPN) was produced.
Cross-reactivity between CHO/MAP-ferPDPN and antiPDPN mAbs, which are useful for immunohistochemistry,
was investigated using flow cytometry. The results demonstrated that PMab-38 for dog PDPN, PMab-52 for cat PDPN,
PMab-237 for whale PDPN, and PMab-241 for bear PDPN,
all cross-reacted with CHO/MAP-ferPDPN cells (Fig. 1). In
contrast, PMab-2 for rat PDPN, PMab-32 for rabbit PDPN,
PMab-44 for bovine PDPN, PMab-48 for dog PDPN, PMab210 for pig PDPN, PMab-213 for pig PDPN, PMab-219 forhorse PDPN, PMab-225 for alpaca PDPN, PMab-231 for tiger PDPN, PMab-233 for Tasmanian devil PDPN, PMab-235
for goat PDPN, PMab-247 for bear PDPN, and PMab-256 forsheep PDPN, did not react with CHO/MAP-ferPDPN cells(Fig. 1). The positive control, anti-MAP tag mAb, reactedwith CHO/MAP-ferPDPN cells as expected.

‰
FIG. 1. Flow cytometry analyses using various anti-PDPN mAbs. Anti-PDPN mAbs (red line) or an anti-MAP tag (redline), or negative control (0.1% BSA in PBS; gray shade) were treated with CHO/MAP-ferPDPN cells at a concentration of
10 mg/mL for 30 minutes at 4C, followed by secondary antibodies. BSA, bovine serum albumin; CHO, Chinese hamster
ovary; ferPDPN, ferret PDPN; PBS, phosphate-buffered saline; PDPN, podoplanin; mAbs, monoclonal antibodies.

136

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

FIG. 2. Immunohistochemical analyses of ferret lung tissues using PMab-241. Histological sections of ferret lung tissues
were incubated with 10 mg/mL of PMab-241 (A–C) or blocking buffer (D–F). (G–I) Hematoxylin and eosin staining using
the serial section of the ferret lung tissues. Scale bar = 100 mm. Red arrows: lymphatic endothelial cells.

FIG. 3. Immunohistochemical analyses of ferret kidney tissues using PMab-241. Histological sections of ferret kidney
tissues were incubated with 10 mg/mL of PMab-241 (A, B) or blocking buffer (C, D). (E, F) Hematoxylin and eosin staining
using the serial section of the ferret kidney tissues. Scale bar = 100 mm.
137

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

138

NANAMIYA ET AL.

Among the four anti-PDPN mAbs that cross-reacted with
CHO/MAP-ferPDPN cells, PMab-241 has demonstrated
having the best sensitivity in immunohistochemical analyses
in our previous study.(37) Therefore, PMab-241 was selected
for all subsequent immunohistochemical analyses of ferret
tissues.

Funding Information

Immunohistochemical analysesof the ferret lung using PMab-241

1. Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P,
Poczewski H, Kalt R, Schaffner G, and Kerjaschki D: Podoplanin, novel 43-kd membrane protein of glomerularepithelial cells, is down-regulated in puromycin nephrosis.
Am J Pathol 1997;151:1141–1152.
2. Douglas YL, Mahtab EA, Jongbloed MR, Uhrin P, Zaujec
J, Binder BR, Schalij MJ, Poelmann RE, Deruiter MC, andGittenberger-de Groot AC: Pulmonary vein, dorsal atrial
wall and atrial septum abnormalities in podoplaninknockout mice with disturbed posterior heart field contribution. Pediatr Res 2009;65:27–32.
3. Ramirez MI, Millien G, Hinds A, Cao Y, Seldin DC, andWilliams MC: T1alpha, a lung type I cell differentiation
gene, is required for normal lung cell proliferation and alveolus formation at birth. Dev Biol 2003;256:61–72.
4. Schacht V, Ramirez MI, Hong YK, Hirakawa S, Feng D,
Harvey N, Williams M, Dvorak AM, Dvorak HF, Oliver G,
and Detmar M: T1alpha/podoplanin deficiency disrupts
normal lymphatic vasculature formation and causes lymphedema. EMBO J 2003;22:3546–3556.
5. Peterziel H, Muller J, Danner A, Barbus S, Liu HK,
Radlwimmer B, Pietsch T, Lichter P, Schutz G, Hess J, andAngel P: Expression of podoplanin in human astrocytic
brain tumors is controlled by the PI3K-AKT-AP-1 signaling pathway and promoter methylation. Neuro Oncol 2012;
14:426–439.
6. Takagi S, Oh-hara T, Sato S, Gong B, Takami M, andFujita N: Expression of Aggrus/podoplanin in bladdercancer and its role in pulmonary metastasis. Int J Cancer
2014;134:2605–2614.
7. Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, and Osawa
M: Enhanced expression of Aggrus (T1alpha/podoplanin),a platelet-aggregation-inducing factor in lung squamouscell carcinoma. Tumor Biol 2005;26:195–200.
8. Suzuki-Inoue K, Kato Y, Inoue O, Kaneko MK, Mishima
K, Yatomi Y, Yamazaki Y, Narimatsu H, and Ozaki Y:
Involvement of the snake toxin receptor CLEC-2, in
podoplanin-mediated platelet activation, by cancer cells.
J Biol Chem 2007;282:25993–26001.
9. Kato Y, Kaneko MK, Kunita A, Ito H, Kameyama A,
Ogasawara S, Matsuura N, Hasegawa Y, Suzuki-Inoue K,
Inoue O, Ozaki Y, and Narimatsu H: Molecular analysis of
the pathophysiological binding of the platelet aggregationinducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 2008;99:54–61.
10. Rayes J, Lax S, Wichaiyo S, Watson SK, Di Y, Lombard S,
Grygielska B, Smith SW, Skordilis K, and Watson SP: The
podoplanin-CLEC-2 axis inhibits inflammation in sepsis.
Nat Commun 2017;8:2239.
11. Bieniasz-Krzywiec P, Martin-Perez R, Ehling M, GarciaCaballero M, Pinioti S, Pretto S, Kroes R, Aldeni C, Di
Matteo M, Prenen H, Tribulatti MV, Campetella O, Smeets
A, Noel A, Floris G, Van Ginderachter JA, and Mazzone
M: Podoplanin-expressing macrophages promote lymphangiogenesis and lymphoinvasion in breast cancer. Cell
Metab 2019;30:917–936 e910.

The expression of ferPDPN in ferret lung using PMab-241
by immunohistochemistry was next evaluated. Our previous
immunohistochemical studies reported that PMab-241 reacts
with lymphatic endothelial cells of the bear lung, but not with
type I alveolar cells.(37) In this study, PMab-241 reacted withlymphatic endothelial cells in the ferret lung, but not with typeI alveolar cells of the ferret lung (Fig. 2A–C). This result issimilar to our previous findings that PMab-241 was also detectable in bear lung.(37) No staining was observed in any of
the serial sections of the ferret lung when stained with thebuffer negative control (Fig. 2D–F). HE staining using theserial sections of the ferret lung is shown in Figure 2G–I.
Immunohistochemical analyses of the ferret kidneyusing PMab-241

We next investigated whether PMab-241 could react withthe ferret kidney in immunohistochemical analyses. Results
demonstrate that PMab-241 stained Bowman’s capsules andglomeruli of the ferret kidney (Fig. 3A, B). The serial sectionof the ferret kidney was not stained by the negative buffer
control (Fig. 3C, D). HE staining using the serial sections wasshown in Figure 3E and F.
Discussion

Ferrets have frequently been used as model species formany human diseases.(42–45) Due to having similar symptoms
to humans when infected with the human influenza virus,
such as runny nose and fever, they are used as animal models
for human influenza preclinical evaluation and influenza
vaccine efficacy studies.(42,43) Ferrets have been also used in
gastritis and gastric adenocarcinoma models, both of which
are related to Helicobacter mustelae infection.(44,45) Recently, ferrets have been utilized as model animals for the
SARS-CoV-2 outbreak.(46,47) Although the wide use of ferrets for these disease models, plus others, specific anti-ferret
mAbs have not yet been developed.
In this study, we have demonstrated that an anti-bear PDPN
mAb, PMab-241, can detect ferPDPN by flow cytometry andimmunohistochemistry. PMab-241 is the first mAb that canstain lymphatic endothelial cells of the ferret lung and glomeruli of the ferret kidney. However, type I alveolar cells in
the ferret lung were not stained by PMab-241 in this study,
indicating that PMab-241 reactivity in ferret exhibits tissue
specificity. These results demonstrate the further need to investigate whether PMab-241 could be utilized for pathophysiological studies using ferrets in the future.
Author Disclosure Statement

No competing financial interests exist.

This research was supported in part by Japan Agency for
Medical Research and Development under Grant Numbers
JP20am0401013 (Y.K.) and JP20am0101078 (Y.K.).
ReferencesDownloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

FERRET PDPN IN IMMUNOHISTOCHEMISTRY

12. Martin-Villar E, Fernandez-Munoz B, Parsons M, Yurrita
MM, Megias D, Perez-Gomez E, Jones GE, and Quintanilla
M: Podoplanin associates with CD44 to promote directional
cell migration. Mol Biol Cell 2010;21:4387–4399.
13. Tsuneki M, Maruyama S, Yamazaki M, Xu B, Essa A, Abe
T, Babkair H, Cheng J, Yamamoto T, and Saku T: Extracellular heat shock protein A9 is a novel interaction partnerof podoplanin in oral squamous cell carcinoma cells. Biochem Biophys Res Commun 2013;434:124–130.
14. Tejchman A, Lamerant-Fayel N, Jacquinet JC, BielawskaPohl A, Mleczko-Sanecka K, Grillon C, Chouaib S,
Ugorski M, and Kieda C: Tumor hypoxia modulates
podoplanin/CCL21 interactions in CCR7+ NK cell recruitment and CCR7+ tumor cell mobilization. Oncotarget
2017;8:31876–31887.
15. Shen Y, Chen CS, Ichikawa H, and Goldberg GS: SRC
induces podoplanin expression to promote cell migration.
J Biol Chem 2010;285:9649–9656.
16. Durchdewald M, Guinea-Viniegra J, Haag D, Riehl A,
Lichter P, Hahn M, Wagner EF, Angel P, and Hess J: Podoplanin is a novel fos target gene in skin carcinogenesis.
Cancer Res 2008;68:6877–6883.
17. Miyashita T, Higuchi Y, Kojima M, Ochiai A, and Ishii G:
Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity ofsquamous cell carcinoma cells. Sci Rep 2017;7:39971.
18. Sikorska J, Gawel D, Domek H, Rudzinska M, and Czarnocka B: Podoplanin (PDPN) affects the invasiveness of
thyroid carcinoma cells by inducing ezrin, radixin andmoesin (E/R/M) phosphorylation in association with matrix
metalloproteinases. BMC Cancer 2019;19:85.
19. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R,
Amann G, Kriehuber E, Diem K, Weninger W, Tschachler
E, Alitalo K, and Kerjaschki D: Angiosarcomas express
mixed endothelial phenotypes of blood and lymphatic
capillaries: Podoplanin as a specific marker for lymphatic
endothelium. Am J Pathol 1999;154:385–394.
20. Kaji C, Tsujimoto Y, Kato Kaneko M, Kato Y, and Sawa
Y: Immunohistochemical examination of novel rat monoclonal antibodies against mouse and human podoplanin.
Acta Histochem Cytochem 2012;45:227–237.
21. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
22. Oki H, Ogasawara S, Kaneko MK, Takagi M, Yamauchi M,
and Kato Y: Characterization of monoclonal antibody
LpMab-3 recognizing sialylated glycopeptide of podoplanin. Monoclon Antib Immunodiagn Immunother 2015;34:
44–50.
23. Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Takagi M,
Kaneko MK, and Kato Y: Development of sensitive
monoclonal antibody PMab-2 against rat podoplanin.
Monoclon Antib Immunodiagn Immunother 2015;34:396–
403.
24. Honma R, Fujii Y, Ogasawara S, Oki H, Liu X, Nakamura
T, Kaneko MK, Takagi M, and Kato Y: Establishment of anovel monoclonal antibody PMab-32 against rabbit podoplanin. Monoclon Antib Immunodiagn Immunother 2016;35:41–47.
25. Honma R, Kaneko MK, Ogasawara S, Fujii Y, Konnai S,
Takagi M, and Kato Y: Specific detection of dog podoplanin
expressed in renal glomerulus by a novel monoclonal antibody PMab-38 in immunohistochemistry. Monoclon AntibImmunodiagn Immunother 2016;35:212–216.

139

26. Yamada S, Itai S, Kaneko MK, and Kato Y: PMab-48
recognizes dog podoplanin of lymphatic endothelial cells.
Monoclon Antib Immunodiagn Immunother 2018;37:
63–66.
27. Honma R, Ogasawara S, Kaneko M, Fujii Y, Oki H,
Nakamura T, Takagi M, Konnai S, and Kato Y: PMab-44
detects bovine podoplanin in immunohistochemistry. Monoclon Antib Immunodiagn Immunother 2016;35:186–190.
28. Yamada S, Itai S, Nakamura T, Yanaka M, Saidoh N,
Chang YW, Handa S, Harada H, Kagawa Y, Ichii O,
Konnai S, Kaneko MK, and Kato Y: PMab-52: Specific andsensitive monoclonal antibody against cat podoplanin for
immunohistochemistry. Monoclon Antib Immunodiagn
Immunother 2017;36:224–230.
29. Furusawa Y, Kaneko MK, Nakamura T, Itai S, Fukui M,
Harada H, Yamada S, and Kato Y: Establishment of a
monoclonal antibody PMab-231 for tiger podoplanin.
Monoclon Antib Immunodiagn Immunother 2019;38:89–95.
30. Furusawa Y, Yamada S, Itai S, Nakamura T, Yanaka M,
Sano M, Harada H, Fukui M, Kaneko MK, and Kato Y:
PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin. Biochem Biophys
Rep 2019;18:100616.
31. Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, andKaneko MK: Anti-horse podoplanin monoclonal antibody
PMab-219 is useful for detecting lymphatic endothelial
cells by immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2018;37:272–274.
32. Kato Y, Yamada S, Furusawa Y, Itai S, Nakamura T, Yanaka M, Sano M, Harada H, Fukui M, and Kaneko MK:
PMab-213: A monoclonal antibody for immunohistochemical analysis against pig podoplanin. Monoclon AntibImmunodiagn Immunother 2019;38:18–24.
33. Furusawa Y, Yamada S, Nakamura T, Sano M, Sayama Y,
Itai S, Takei J, Harada H, Fukui M, Kaneko MK, and Kato
Y: PMab-235: A monoclonal antibody for immunohistochemical analysis against goat podoplanin. Heliyon 2019;5:e02063.
34. Kato Y, Furusawa Y, Yamada S, Itai S, Takei J, Sano M,
and Kaneko MK: Establishment of a monoclonal antibody
PMab-225 against alpaca podoplanin for immunohistochemical analyses. Biochem Biophys Rep 2019;18:
100633.
35. Furusawa Y, Yamada S, Itai S, Nakamura T, Takei J, Sano
M, Harada H, Fukui M, Kaneko MK, and Kato Y: Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil
podoplanin. Biochem Biophys Rep 2019;18:100631.
36. Furusawa Y, Takei J, Sayama Y, Yamada S, Kaneko MK,
and Kato Y: Development of an anti-bear podoplanin
monoclonal antibody PMab-247 for immunohistochemical
analysis. Biochem Biophys Rep 2019;18:100644.
37. Takei J, Yamada S, Konnai S, Ishinazaka T, Shimozuru M,
Kaneko MK, and Kato Y: PMab-241 specifically detects
bear podoplanin of lymphatic endothelial cells in the lungof brown bear. Monoclon Antib Immunodiagn Immunother2019;38:282–284.
38. Kato Y, Furusawa Y, Itai S, Takei J, Nakamura T, Sano M,
Harada H, Yamada S, and Kaneko MK: Establishment ofan anticetacean podoplanin monoclonal antibody PMab237 for immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2019;38:108–113.
39. Kato Y, Furusawa Y, Sano M, Takei J, Nakamura T,
Yanaka M, Okamoto S, Handa S, Komatsu Y, Asano T,

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

140

Sayama Y, and Kaneko MK: Development of an anti-sheep
podoplanin monoclonal antibody PMab-256 for immunohistochemical analysis of lymphatic endothelial cells.
Monoclon Antib Immunodiagn Immunother 2020;39:82–90.
40. Fujii Y, Kaneko MK, and Kato Y: MAP tag: A novel tagging system for protein purification and detection. Monoclon
Antib Immunodiagn Immunother 2016;35:293–299.
41. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T,
Yanaka M, Fukui M, Harada H, Mizuno T, Sakai Y, Takasu
M, Kaneko MK, and Kato Y: PMab-210: A monoclonal
antibody against pig podoplanin. Monoclon Antib Immunodiagn Immunother 2019;38:30–36.
42. Belser JA, Katz JM, and Tumpey TM: The ferret as amodel organism to study influenza A virus infection. Dis
Model Mech 2011;4:575–579.
43. Matsuoka Y, Lamirande EW, and Subbarao K: The ferretmodel for influenza. Curr Protoc Microbiol 2009;Chapter
15:Unit 15G 12.
44. Fox JG, Otto G, Taylor NS, Rosenblad W, and Murphy JC:
Helicobacter mustelae-induced gastritis and elevated gastric pH in the ferret (Mustela putorius furo). Infect Immun
1991;59:1875–1880.
45. Fox JG, Dangler CA, Sager W, Borkowski R, and Gliatto
JM: Helicobacter mustelae-associated gastric adenocarcinoma in ferrets (Mustela putorius furo). Vet Pathol 1997;
34:225–229.

NANAMIYA ET AL.

46. Kim YI, Kim SG, Kim SM, Kim EH, Park SJ, Yu KM,
Chang JH, Kim EJ, Lee S, Casel MAB, Um J, Song MS,
Jeong HW, Lai VD, Kim Y, Chin BS, Park JS, Chung KH,
Foo SS, Poo H, Mo IP, Lee OJ, Webby RJ, Jung JU, andChoi YK: Infection and rapid transmission of SARS-CoV-2
in ferrets. Cell Host Microbe 2020;27:704–709 e702.
47. Shi J, Wen Z, Zhong G, Yang H, Wang C, Huang B, Liu R,
He X, Shuai L, Sun Z, Zhao Y, Liu P, Liang L, Cui P,
Wang J, Zhang X, Guan Y, Tan W, Wu G, Chen H, and Bu
Z: Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2. Science 2020;368:
1016–1020.

Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 31, 2021
Accepted: May 4, 2021