MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 2, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0067
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Development of a Monoclonal Antibody
PMab-292 Against Ferret Podoplanin

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Nohara Goto,1 Hiroyuki Suzuki,1 Tomohiro Tanaka,2 Teizo Asano,2 Mika K. Kaneko,2 and Yukinari Kato1,2

Ferrets (Mustela putorius furo) have been used as small animal models to investigate severe acute respiratory
syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infections. Pathological analyses of these tissue
samples, including those of the lung, are, therefore, essential to understand the pathogenesis of SARS-CoVs andevaluate the action of therapeutic monoclonal antibodies (mAbs) against this disease. However, mAbs thatrecognize ferret-derived proteins and distinguish between specific cell types, such as lung epithelial cells, arelimited. Podoplanin (PDPN) has been identified as an essential marker in lung type I alveolar epithelial cells,
kidney podocytes, and lymphatic endothelial cells. In this study, an anti-ferret PDPN (ferPDPN) mAb PMab292 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method.
PMab-292 recognized ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells by flow cytometry and Western blotting. The kinetic analysis using flow cytometry showed that the KD of PMab-292 for
CHO/ferPDPN was 3.4 · 10-8 M. Furthermore, PMab-292 detected lung type I alveolar epithelial cells, lymphatic endothelial cells, and glomerular/Bowman’s capsule in the kidney using immunohistochemistry. Hence,these results propose the usefulness of PMab-292 in analyzing ferret-derived tissues for SARS-CoV-2 research.
Keywords: ferret podoplanin, monoclonal antibody, CBIS, immunohistochemistry

Introduction

F

errets (Mustela putorius furo) have been used as ahighly valuable model for testing the pathogenicity andtransmission of human respiratory viruses, including the influenza virus,(1) severe acute respiratory syndrome coronavirus (SARS-CoV),(2) and SARS-CoV-2.(3) Aftermucosal exposure to SARS-CoV-2, clinical alterations inferrets have been identified as undetectable or mild. In addition, they can transmit viruses efficiently to uninfected
ferrets through direct contact and aerosols.(4,5) Similar to thegolden (Syrian) hamster (Mesocricetus auratus) model, viral
replication has also been detected in the upper respiratory
tract of ferrets after a 2-week infection period. However, incontrast to the golden hamster,(6) reductions in body weight
are absent or minimal.
In histopathological analysis, inflammation within alveolar spaces and perivascular mononuclear parts were observedin SARS-CoV-2-infected ferrets. In addition, the bronchialsubmucosal foci with eosinophilic materials and collagen
degeneration were observed. Microscopic observations of
SARS-CoV-2-infected ferrets, therefore, suggested mild
bronchoalveolar or alveolar inflammation.(3) These studies
also indicated that airborne transmission of SARS-CoV-2 canoccur, thereby suggesting the usefulness of the ferret modelin SARS-CoV-2 research.(7) However, there is a limitation of
the pathological analysis owing to the lack of antibodies thatcan recognize the ferret-derived antigens and distinguish thespecific cells in the lung.
Podoplanin (PDPN) is a type I transmembrane mucin-likesialoglycoprotein and an important marker in lung type I
alveolar epithelial cells,(8,9) kidney podocytes,(10) and lymphatic endothelial cells.(11,12) Therefore, anti-ferret PDPN
(ferPDPN) monoclonal antibodies (mAbs) have been proposed as a useful tool to investigate the pathogenesis of lungtype I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells in SARS-CoV-2 infection models.
This study developed anti-ferPDPN mAbs using the Cell-

Departments of 1Molecular Pharmacology and 2Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Japan.

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Based Immunization and Screening (CBIS) method. The
CBIS method includes the immunization of antigenoverexpressed cells and subsequent high-throughput hybridoma screening through flow cytometry. We establishedan anti-ferPDPN mAb PMab-292 and investigated the several applications, including flow cytometry, Western blotting, and immunohistochemistry.
Materials and Methods

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Cell lines

Chinese hamster ovary (CHO)-K1 and mouse multiple
myeloma P3X63Ag8U.1 (P3U1) cells were obtained fromthe American Type Culture Collection (ATCC; Manassas,
VA). Synthesized DNA (Eurofins Genomics KK, Tokyo,
Japan) encoding ferPDPN plus an N-terminal MAP tag(GDGMVPPGIEDK)(13,14) and an N-terminal 2 · RIEDL tag(RIEDLRIEDL),(15–20) which are recognized by an antiMAP tag mAb (PMab-1) and an anti-RIEDL tag mAb(LpMab-7), respectively, were subcloned into a pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan). Afterward, plasmids were transfected into CHO-K1
cells, using Lipofectamine LTX with Plus Reagent (Thermo
Fisher Scientific, Inc., Waltham, MA). Stable transfectants(CHO/MAP-ferPDPN and CHO/2 · RIEDL-ferPDPN) were
subsequently selected using cell sorter (SH800; Sony Corp.,
Tokyo, Japan).
Next, CHO-K1, CHO/MAP-ferPDPN, CHO/2 · RIEDLferPDPN, and P3U1 were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc.,
Kyoto, Japan), supplemented with 10% heat-inactivatedfetal bovine serum (Thermo Fisher Scientific, Inc.),
100 U/mL penicillin, 100 lg/mL streptomycin, and0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.). CHO/
MAP-ferPDPN was cultivated in a medium containing
0.5 mg/mL Zeocin (InvivoGen, San Diego, CA). Cells werethen cultured in an incubator at 37C humidity, 5% CO2, and
95% air atmosphere.
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. In this study, we used a CBIS method(21–27) todevelop mAbs against ferPDPN. One BALB/c mouse wassubsequently immunized intraperitoneally (i.p.) with CHO/
MAP-ferPDPN cells (1 · 108) using Imject Alum (Thermo
Fisher Scientific, Inc.). The procedure included three additional immunization with CHO/MAP-ferPDPN cells(1 · 108), followed by a final booster injection of CHO/MAPferPDPN cells (1 · 108) 2 days before harvesting splenic
cells.
Subsequently, splenic cells were fused with P3U1 cells
using polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in
RPMI 1640 media supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific, Inc.), after which culture supernatants were screened
through flow cytometry for anti-ferPDPN antibody production.

GOTO ET AL.
Flow cytometry

Cells (2 · 105 cells/mL) were harvested after brief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic
acid (Nacalai Tesque, Inc.). After washing with 0.1% bovine
serum albumin (Nacalai Tesque, Inc.) in phosphate-bufferedsaline (PBS; Nacalai Tesque, Inc.), cells were treated with
PMab-292 (1–0.001 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 collected using SA3800
Cell Analyzer (Sony Biotechnology Corp.).
Determining of the binding affinity

Cells were suspended in 100 lL serially diluted PMab-292(10–0.0006 mg/mL), followed by a subsequent suspension inan Alexa Fluor 488-conjugated anti-mouse IgG solution(1:200; Cell Signaling Technology, Inc.). Afterward, fluorescence data 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.
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) 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-292,
an anti-MAP tag mAb (PMab-1), or an anti-b-actin mAb(clone AC-15; Sigma-Aldrich Corp., St. Louis, MO). Then,they were incubated again with peroxidase-conjugated antimouse immunoglobulins (diluted 1:1000; Agilent Technologies, Inc., Santa Clara, CA). Finally, protein bands weredetected using ImmunoStar LD (FUJIFILM Wako Pure
Chemical Corporation) and a Sayaca-Imager (DRC Co. Ltd.,Tokyo, Japan).
Immunohistochemical analysis

Ferret tissue samples were collected after autopsy procedures were completed at the Yamaguchi University, and fixedin 10% neutral-buffered formalin,(28) after which paraffinembedded tissue sections (4 lm thick) were made. Subsequently, histological sections (4 lm thick) of ferret tissue
samples were directly autoclaved in a citrate buffer (pH 6.0;
Nichirei Biosciences, Inc., Tokyo, Japan) for 20 minutes.
After blocking histological sections with a SuperBlock T20(PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.),
sections were incubated with PMab-292 (5 lg/mL) for 1 hourat room temperature and treated with the EnVision+Kit formouse (Agilent Technologies, Inc.) for 30 minutes.
Color was developed with 3,3¢-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes,
after counterstaining using hematoxylin (FUJIFILM Wako
Pure Chemical Corporation). Hematoxylin and eosin (FUJIFILM Wako Pure Chemical Corporation) staining was also
conducted using the serial section of ferret tissue samples.

DEVELOPMENT OF ANTI-FERRET PDPN mAb
Results
Establishment of anti-ferPDPN mAbs

To develop anti-ferPDPN mAbs, the CBIS method, using
stable transfectants for immunization and flow cytometry,
was used (Fig. 1). CHO/MAP-ferPDPN cells, which overexpressed ferPDPN and had an N-terminal MAP tag (alsoreferred to as CHO/ferPDPN) were immunized into one
mouse. Hybridomas were then seeded into 96-well plates,
and CHO/2 · RIEDL-ferPDPN-positive and CHO-K1negative wells were identified. After conducting limiting
dilution and selection using immunohistochemistry, thePMab-292 (mouse IgG1, kappa) was finally isolated.

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cytometry. As indicated in Figure 2B, the KD for PMab-292
interactions with CHO/ferPDPN cells was 3.4 · 10-8 M,
suggesting that PMab-292 exhibited a moderate affinity for
ferPDPN.
Western blotting

Western blotting was conducted to further assess the sensitivity of PMab-292. For this assessment, lysates of CHO-K1
and CHO/ferPDPN cells were probed. As demonstrated in
Figure 3, PMab-292 detected the 48-kDa band of ferPDPN in
lysates from CHO/ferPDPN cells, whereas this band wasabsent in lysates from CHO-K1 cells, indicating that PMab292 specifically detected ferPDPN.

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Flow cytometric analyses

Flow cytometric analyses were conducted using PMab292 with CHO/ferPDPN and CHO-K1 cells. PMab-292 recognized CHO/ferPDPN, but did not react with CHO-K1 cells(Fig. 2A). The kinetic analysis of PMab-292 interactions with
CHO/ferPDPN cells was, therefore, conducted using flow

Immunohistochemical analyses

To investigate whether PMab-292 can be used for immunohistochemical analyses using formalin-fixed paraffinembedded (FFPE) ferret tissue sections, normal lung andkidney tissue samples from ferrets were examined. Both

FIG. 1. Schematic procedure showing the CBIS method. CHO/ferPDPN cells were inoculated i.p. into the mice. Splenic
cells were then fused with myeloma cells, after which culture supernatants from hybridomas were screened for antiferPDPN antibody production using flow cytometry. CBIS, Cell-Based Immunization and Screening; i.p., intraperitoneally.

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GOTO ET AL.

FIG. 2. Flow cytometry using anti-ferPDPN mAb, PMab-292. (A) CHO-K1 and CHO/ferPDPN were treated with 1–
0.001 mg/mL of PMab-292, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG. The black line
represents the negative control. (B) CHO/ferPDPN cells were suspended in 100 mL serially diluted PMab-292 (10–0.0006 mg/mL), after subsequent treatment with Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were
subsequently collected using BD FACSLyric, after which we calculated the dissociation constant (KD) using GraphPad
PRISM 8. mAb, monoclonal antibody.
tissue samples expressed PDPN in different species, including human,(29) mouse,(30) rat,(31) rabbit,(32) dog,(33) cat,(34)cow,(35) pig,(36) Tasmanian devil,(37) alpaca,(38) tiger,(39)whale,(40) goat,(41) horse,(42) bears,(43) and sheep.(24,44) As indicated in Figure 4, PMab-292 strongly and specifically identified type I alveolar cell within the lung. Lymphatic
endothelial cells of the lung were also detected using PMab292 (Fig. 4C). Moreover, PMab-292 was clearly stained in theglomerulus and Bowman’s capsule in the kidney (Fig. 5A, B).
Therefore, these results indicated the usefulness of PMab-292
for detecting ferPDPN-positive cells in FFPE tissue samples.
Discussion

Previously, we reported the cross-reactivity of anti-bear
PDPN mAb (PMab-241) with ferret PDPN-overexpressed cells
and ferret tissue samples.(28) Through flow cytometry, PMab241 recognized ferret PDPN-overexpressed CKO-K1 cells.
PMab-241 also reacted with lymphatic endothelial cells, but notwith type I alveolar epithelial cells in the ferret lung. In thisstudy, we established a novel anti-ferret PDPN mAb, PMab-292,
which is useful for flow cytometry and immunohistochemical
staining. Especially, PMab-292 stained type I alveolar epithelial
cells of the ferret lung (Fig. 4). Therefore, PMab-292 can be used
to detect and analyze the development and/or pathogenesis oftype I alveolar epithelial cells. Previously, we have conducted aconventional alanine-scanning method(16,24,45–65) and the REMAP method(17–20) to determine conformational epitopes ofmAbs. Further studies are also warranted to determine the epitope of PMab-292 using those methods.
Ferrets have been used as animal models for investigating
the pathogenicity and transmission of both SARS-CoV and
SARS-CoV-2. Therefore, we can compare the pathogenesisof viral-based respiratory tract infections. SARS-CoV infections demonstrated bronchial and bronchiolar hyperplasia,
and perivascular cuffing in ferret lung tissue samples. However, no evidence of enhanced disease was observed in any
ferret.(66) With SARS-CoV-2 infections in ferrets, several
reports proposed that the clinical disease remained mildcompared with the golden hamsters.(67)Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/22. For personal use only.

FIG. 3. Western blotting using PMab-292. Cell lysates (10 mg) of CHO-K1 and CHO/ferPDPN cells were electrophoresed, after which proteins were transferred to PVDF membranes. After blocking, membranes were subsequently
incubated with 1 mg/mL PMab-292, anti-MAP tag mAb (PMab-1), and anti-b-actin, followed by incubation withperoxidase-conjugated anti-mouse immunoglobulins. PVDF, polyvinylidene difluoride.

FIG. 4. Immunohistochemical analysis for the ferret lung. Histological sections of the ferret lung were directly autoclavedin a citrate buffer and incubated with 5 mg/mL of PMab-292 (A–C) or a blocking buffer (D–F), followed by the EnVision+Kit. (G–I) H&E staining was performed. Arrows show that PDPN was expressed in lymphatic endothelial cells.
Scale bars = 100 mm. H&E, hematoxylin and eosin.
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GOTO ET AL.

FIG. 5. Immunohistochemical analysis for the ferret kidney. Histological sections from the ferret kidney were directly
autoclaved in a citrate buffer and incubated with 5 mg/mL of PMab-292 (A, B) or blocking buffer (C, D), followed by the
EnVision+Kit. (E, F) H&E staining. Scale bars = 100 mm.
Nevertheless, a persistent inflammation in the nasal turbinates was observed in young ferrets, after which follicular
hyperplasia in the bronchi developed 21-day postinfection.(68) Therefore, the ferret model of SARS-CoV-2 infection resembles human asymptomatic infection.(69) The use of
PMab-292 to investigate the morphological alterations inlung type I alveolar epithelial cells is also proposed to provide
valuable information on the pathogenesis of viral infections,
thereby contributing to the development of drugs against
these infections, including the SARS-CoV-2.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under Grant
Nos. JP21am0401013 and JP21am0101078 (to Y.K.).
References

1. Nguyen TQ, Rollon R, and Choi YK: Animal models for
influenza research: Strengths and weaknesses. Viruses
2021;13:1011.
2. van den Brand JM, Haagmans BL, van Riel D, Osterhaus
AD, and Kuiken T: The pathology and pathogenesis ofexperimental severe acute respiratory syndrome and influenza in animal models. J Comp Pathol 2014;151:83–
112.
3. Muñoz-Fontela C, Dowling WE, Funnell SGP, Gsell PS,
Riveros-Balta AX, Albrecht RA, Andersen H, Baric RS,
Carroll MW, Cavaleri M, Qin C, Crozier I, Dallmeier K, deWaal L, de Wit E, Delang L, Dohm E, Duprex WP, Falzarano D, Finch CL, Frieman MB, Graham BS, Gralinski
LE, Guilfoyle K, Haagmans BL, Hamilton GA, Hartman
AL, Herfst S, Kaptein SJF, Klimstra WB, Knezevic I,
Krause PR, Kuhn JH, Le Grand R, Lewis MG, Liu WC,
Maisonnasse P, McElroy AK, Munster V, Oreshkova N,
Rasmussen AL, Rocha-Pereira J, Rockx B, Rodrı́guez E,
Rogers TF, Salguero FJ, Schotsaert M, Stittelaar KJ, Thibaut HJ, Tseng CT, Vergara-Alert J, Beer M, Brasel T,
Chan JFW, Garcı́a-Sastre A, Neyts J, Perlman S, Reed DS,
Richt JA, Roy CJ, Segalés J, Vasan SS, Henao-Restrepo
AM, and Barouch DH: Animal models for COVID-19.
Nature 2020;586:509–515.
4. 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.
5. 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Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/22. For personal use only.

DEVELOPMENT OF ANTI-FERRET PDPN mAb

Bu Z: Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2. Science 2020;
368:1016–1020.
6. Sia SF, Yan LM, Chin AWH, Fung K, Choy KT, Wong
AYL, Kaewpreedee P, Perera R, Poon LLM, Nicholls JM,
Peiris M, and Yen HL: Pathogenesis and transmission of
SARS-CoV-2 in golden hamsters. Nature 2020;583:834–
838.
7. Richard M, Kok A, de Meulder D, Bestebroer TM, Lamers
MM, Okba NMA, Fentener van Vlissingen M, Rockx B,
Haagmans BL, Koopmans MPG, Fouchier RAM, andHerfst S: SARS-CoV-2 is transmitted via contact and via
the air between ferrets. Nat Commun 2020;11:3496.
8. Dobbs LG, Williams MC, and Gonzalez R: Monoclonal
antibodies specific to apical surfaces of rat alveolar type I
cells bind to surfaces of cultured, but not freshly isolated,
type II cells. Biochim Biophys Acta 1988;970:146–156.
9. Rishi AK, Joyce-Brady M, Fisher J, Dobbs LG, Floros J,
VanderSpek J, Brody JS, and Williams MC: Cloning,
characterization, and development expression of a rat lungalveolar type I cell gene in embryonic endodermal andneural derivatives. Dev Biol 1995;167:294–306.
10. 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.
11. Hirakawa S, Hong YK, Harvey N, Schacht V, Matsuda K,
Libermann T, and Detmar M: Identification of vascularlineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.
Am J Pathol 2003;162:575–586.
12. Petrova TV, Mäkinen T, Mäkelä TP, Saarela J, Virtanen I,
Ferrell RE, Finegold DN, Kerjaschki D, Ylä-Herttuala S,
and Alitalo K: Lymphatic endothelial reprogramming ofvascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J 2002;21:4593–4599.
13. 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.
14. Wakasa A, Kaneko MK, Kato Y, Takagi J, and Arimori T:
Site-specific epitope insertion into recombinant proteinsusing the MAP tag system. J Biochem 2020;168:375–384.
15. Asano T, Kaneko MK, and Kato Y: RIEDL tag: A novel
pentapeptide tagging system for transmembrane protein
purification. Biochem Biophys Rep 2020;23:100780.
16. Sayama Y, Sano M, Kaneko MK, and Kato Y: Epitope
analysis of an anti-whale podoplanin monoclonal antibody,PMab-237, using flow cytometry. Monoclon Antib Immunodiagn Immunother 2020;39:17–22.
17. Sano M, Kaneko MK, Aasano T, and Kato Y: Epitope
mapping of an antihuman EGFR monoclonal antibody(EMab-134) using the REMAP method. Monoclon AntibImmunodiagn Immunother 2021;40:191–195.
18. Asano T, Kaneko MK, Takei J, Tateyama N, and Kato Y:
Epitope mapping of the anti-CD44 monoclonal antibody(C44Mab-46) using the REMAP method. Monoclon AntibImmunodiagn Immunother 2021;40:156–161.
19. Asano T, Kaneko MK, and Kato Y: Development of anovel epitope mapping system: RIEDL insertion for epitope mapping method. Monoclon Antib Immunodiagn Immunother 2021;40:162–167.

107

20. Nanamiya R, Sano M, Asano T, Yanaka M, Nakamura T,
Saito M, Tanaka T, Hosono H, Tateyama N, Kaneko MK,
and Kato Y: Epitope mapping of an anti-human epidermal
growth factor receptor monoclonal antibody (EMab-51)
using the RIEDL insertion for epitope mapping method.
Monoclon Antib Immunodiagn Immunother 2021;40:149–
155.
21. Itai S, Fujii Y, Nakamura T, Chang YW, Yanaka M, Saidoh
N, Handa S, Suzuki H, Harada H, Yamada S, Kaneko MK,
and Kato Y: Establishment of CMab-43, a sensitive andspecific anti-CD133 monoclonal antibody, for immunohistochemistry. Monoclon Antib Immunodiagn Immunother2017;36:231–235.
22. Furusawa Y, Kaneko MK, and Kato Y: Establishment of ananti-CD20 monoclonal antibody (C(20)Mab-60) for immunohistochemical analyses. Monoclon Antib Immunodiagn Immunother 2020;39:112–116.
23. Furusawa Y, Kaneko MK, and Kato Y: Establishment ofC(20)Mab-11, a novel anti-CD20 monoclonal antibody, forthe detection of B cells. Oncol Lett 2020;20:1961–1967.
24. Kaneko MK, Sano M, Takei J, Asano T, Sayama Y, Hosono H, Kobayashi A, Konnai S, and Kato Y: Development
and characterization of anti-sheep podoplanin monoclonal
antibodies PMab-253 and PMab-260. Monoclon AntibImmunodiagn Immunother 2020;39:144–155.
25. Tanaka T, Asano T, Sano M, Takei J, Hosono H, Nanamiya
R, Nakamura T, Yanaka M, Harada H, Fukui M, Suzuki H,
Uchida K, Nakagawa T, Kato Y, and Kaneko MK: Development of monoclonal antibody PMab-269 against California Sea Lion podoplanin. Monoclon AntibImmunodiagn Immunother 2021;40:124–133.
26. Yamada S, Itai S, Nakamura T, Yanaka M, Chang YW,
Suzuki H, Kaneko MK, and Kato Y: Monoclonal antibody
L1Mab-13 detected human PD-L1 in lung cancers. Monoclon Antib Immunodiagn Immunother 2018;37:110–115.
27. Yamada S, Itai S, Nakamura T, Yanaka M, Kaneko MK,
and Kato Y: Detection of high CD44 expression in oral
cancers using the novel monoclonal antibody, C44Mab-5.
Biochem Biophys Rep 2018;14:64–68.
28. Nanamiya R, Takei J, Asano T, Sano M, Tanaka T, Hosono
H, Harada H, Sakai Y, Mizuno T, Suzuki H, Kaneko MK,
and Kato Y: Ferret podoplanin is detected by PMab-241 in
immunohistochemistry. Monoclon Antib Immunodiagn
Immunother 2021;40:134–140.
29. Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano K,
Chiba Y, Hasegawa Y, Hirabayashi J, Narimatsu H, Mishima K, and Osawa M: Inhibition of tumor cell-induced
platelet aggregation using a novel anti-podoplanin antibody
reacting with its platelet-aggregation-stimulating domain.
Biochem Biophys Res Commun 2006;349:1301–1307.
30. 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.
31. 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.
32. 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.

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/22. For personal use only.

108

33. 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 Antib Immunodiagn Immunother 2016;35:212–
216.
34. 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.
35. 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.
36. 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.
37. 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.
38. 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.
39. 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.
40. 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.
41. 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.
42. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T,
Yanaka M, Handa S, Mizuno T, Maeda K, Fukui M, Harada H, Kaneko MK, and Kato Y: Establishment of monoclonal antibody PMab-202 against horse podoplanin.
Monoclon Antib Immunodiagn Immunother 2018;37:233–
237.
43. Takei J, Furusawa Y, Yamada S, Nakamura T, Sayama Y,
Sano M, Konnai S, Kobayashi A, Harada H, Kaneko MK,
and Kato Y: PMab-247 detects bear podoplanin in immunohistochemical analysis. Monoclon Antib Immunodiagn
Immunother 2019;38:171–174.
44. Kato Y, Furusawa Y, Sano M, Takei J, Nakamura T, Yanaka M, Okamoto S, Handa S, Komatsu Y, Asano T,
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.

GOTO ET AL.

45. Chang YW, Kaneko MK, Yamada S, and Kato Y: Epitope
mapping of monoclonal antibody PMab-52 against cat
podoplanin. Monoclon Antib Immunodiagn Immunother2018;37:95–99.
46. Chang YW, Yamada S, Kaneko MK, and Kato Y: Epitope
mapping of monoclonal antibody PMab-38 against dog
podoplanin. Monoclon Antib Immunodiagn Immunother2017;36:291–295.
47. Furusawa Y, Yamada S, Itai S, Nakamura T, Fukui M, Harada
H, Kaneko MK, and Kato Y: Elucidation of critical epitope ofanti-rat podoplanin monoclonal antibody PMab-2. Monoclon
Antib Immunodiagn Immunother 2018;37:188–193.
48. Honma R, Fujii Y, Ogasawara S, Oki H, Konnai S, Kagawa
Y, Takagi M, Kaneko MK, and Kato Y: Critical epitope ofanti-rabbit podoplanin monoclonal antibodies for immunohistochemical analysis. Monoclon Antib Immunodiagn
Immunother 2016;35:65–72.
49. Kaneko MK, Furusawa Y, Sano M, Itai S, Takei J, Harada
H, Fukui M, Yamada S, and Kato Y: Epitope mapping of theantihorse podoplanin monoclonal antibody PMab-202.
Monoclon Antib Immunodiagn Immunother 2019;38:79–84.
50. Kaneko MK, Nakamura T, Kunita A, Fukayama M, Abe S,
Nishioka Y, Yamada S, Yanaka M, Saidoh N, Yoshida K,
Fujii Y, Ogasawara S, and Kato Y: ChLpMab-23: Cancerspecific human-mouse chimeric anti-podoplanin antibody
exhibits antitumor activity via antibody-dependent cellularcytotoxicity. Monoclon Antib Immunodiagn Immunother2017;36:104–112.
51. Kaneko MK, Oki H, Hozumi Y, Liu X, Ogasawara S,
Takagi M, Goto K, and Kato Y: Monoclonal antibody
LpMab-9 recognizes O-glycosylated N-terminus of human
podoplanin. Monoclon Antib Immunodiagn Immunother2015;34:310–317.
52. Kaneko MK, Yamada S, Itai S, Chang YW, Nakamura T,
Yanaka M, and Kato Y: Elucidation of the critical epitopeof an anti-EGFR monoclonal antibody EMab-134. Biochem Biophys Rep 2018;14:54–57.
53. Kato Y, Kunita A, Fukayama M, Abe S, Nishioka Y,
Uchida H, Tahara H, Yamada S, Yanaka M, Nakamura T,
Saidoh N, Yoshida K, Fujii Y, Honma R, Takagi M,
Ogasawara S, Murata T, and Kaneko MK: Antiglycopeptide mouse monoclonal antibody LpMab-21 exerts antitumor activity against human podoplanin through antibodydependent cellular cytotoxicity and complement-dependent
cytotoxicity. Monoclon Antib Immunodiagn Immunother2017;36:20–24.
54. Kato Y, Ogasawara S, Oki H, Goichberg P, Honma R, Fujii
Y, and Kaneko MK: LpMab-12 established by CasMab
technology specifically detects sialylated O-Glycan on
Thr52 of platelet aggregation-stimulating domain of human
podoplanin. PLoS One 2016;11:e0152912.
55. Kato Y, Sayama Y, Sano M, and Kaneko MK: Epitope
analysis of an antihorse podoplanin monoclonal antibody
PMab-219. Monoclon Antib Immunodiagn Immunother2019;38:266–270.
56. Kato Y, Takei J, Furusawa Y, Sayama Y, Sano M, Konnai
S, Kobayashi A, Harada H, Takahashi M, Suzuki H, Yamada S, and Kaneko MK: Epitope mapping of anti-bear
podoplanin monoclonal antibody PMab-247. Monoclon
Antib Immunodiagn Immunother 2019;38:230–233.
57. Sano M, Kaneko MK, and Kato Y: Epitope mapping of
monoclonal antibody PMab-233 against Tasmanian devil
podoplanin. Monoclon Antib Immunodiagn Immunother2019;38:261–265.

Downloaded by Yukinari Kato from www.liebertpub.com at 04/26/22. For personal use only.

DEVELOPMENT OF ANTI-FERRET PDPN mAb

58. Sayama Y, Sano M, Asano T, Furusawa Y, Takei J, Nakamura T, Yanaka M, Okamoto S, Handa S, Komatsu Y,
Nakamura Y, Yanagawa M, Kaneko MK, and Kato Y:
Epitope mapping of PMab-241, a lymphatic endothelial cellspecific anti-bear podoplanin monoclonal antibody. Monoclon Antib Immunodiagn Immunother 2020;39:77–81.
59. Sayama Y, Sano M, Furusawa Y, Kaneko MK, and Kato Y:
Epitope mapping of PMab-225 an anti-alpaca podoplanin
monoclonal antibody using flow cytometry. Monoclon
Antib Immunodiagn Immunother 2019;38:255–260.
60. Takei J, Itai S, Furusawa Y, Yamada S, Nakamura T, Sano
M, Harada H, Fukui M, Kaneko MK, and Kato Y: Epitope
mapping of anti-tiger podoplanin monoclonal antibody
PMab-231. Monoclon Antib Immunodiagn Immunother2019;38:129–132.
61. Tanaka T, Asano T, Sano M, Takei J, Hosono H, Nanamiya R,
Tateyama N, Kaneko MK, and Kato Y: Epitope mapping of theanti-California Sea Lion podoplanin monoclonal antibody
PMab-269 using alanine-scanning mutagenesis and ELISA.
Monoclon Antib Immunodiagn Immunother 2021;40:196–200.
62. Yamada S, Itai S, Furusawa Y, Kaneko MK, and Kato Y:
Epitope mapping of antipig podoplanin monoclonal antibody PMab-213. Monoclon Antib Immunodiagn Immunother 2019;38:224–229.
63. Yamada S, Itai S, Kaneko MK, Konnai S, and Kato Y:
Epitope mapping of anti-mouse podoplanin monoclonal
antibody PMab-1. Biochem Biophys Rep 2018;15:52–56.
64. Yamada S, Kaneko MK, Itai S, Chang YW, Nakamura T,
Yanaka M, Ogasawara S, Murata T, Uchida H, Tahara H,
Harada H, and Kato Y: Epitope mapping of monoclonal
antibody PMab-48 against dog podoplanin. Monoclon
Antib Immunodiagn Immunother 2018;37:162–165.
65. Takei J, Asano T, Suzuki H, Kaneko MK, and Kato Y:
Epitope mapping of the anti-CD44 monoclonal antibody(C44Mab-46) using alanine-scanning mutagenesis andsurface plasmon resonance. Monoclon Antib Immunodiagn
Immunother 2021;40:219–226.

109

66. Darnell ME, Plant EP, Watanabe H, Byrum R, St Claire M,
Ward JM, and Taylor DR: Severe acute respiratory syndrome coronavirus infection in vaccinated ferrets. J Infect
Dis 2007;196:1329–1338.
67. Monchatre-Leroy E, Lesellier S, Wasniewski M, PicardMeyer E, Richomme C, Boué F, Lacôte S, Murri S, Pulido
C, Vulin J, Salguero FJ, Gouilh MA, Servat A, and Marianneau P: Hamster and ferret experimental infection withintranasal low dose of a single strain of SARS-CoV-2.
J Gen Virol 2021;102:001567.
68. van de Ven K, van Dijken H, Wijsman L, Gomersbach A,
Schouten T, Kool J, Lenz S, Roholl P, Meijer A, vanKasteren PB, and de Jonge J: Pathology and immunity after
SARS-CoV-2 infection in male ferrets is affected by age
and inoculation route. Front Immunol 2021;12:750229.
69. Everett HE, Lean FZX, Byrne AMP, van Diemen PM,
Rhodes S, James J, Mollett B, Coward VJ, Skinner P,
Warren CJ, Bewley KR, Watson S, Hurley S, Ryan KA,
Hall Y, Simmons H, Núñez A, Carroll MW, Brown IH, andBrookes SM: Intranasal infection of ferrets with SARSCoV-2 as a model for asymptomatic human infection.
Viruses 2021;13:113.

Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: December 24, 2021
Accepted: January 25, 2022