Biochemistry and Biophysics Reports 24 (2020) 100808

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

Epitope mapping of an anti-diacylglycerol kinase delta monoclonal
antibody DdMab-1
Masato Sano a, Teizo Asano a, Mika K. Kaneko a, Yukinari Kato a, b, *ab

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapanNew Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapanA R T I C L E I N F O

A B S T R A C T

Keywords:
DGKd
DdMab-1
Monoclonal antibody

Diacylglycerol kinase δ (DGKδ) is a type II DGK, which catalyzes diacylglycerol phosphorylation to produce
phosphatidic acid. DGKδ is expressed in several types of tissues and organs including the stomach, testis, bonemarrow, and lymph node. Here, we established an anti-human DGKδ (hDGKδ) mAb, DdMab-1 (mouse IgG2a,kappa), which is useful for Western blot analysis. We also introduced deletion or point mutations to hDGKδ, andperformed western blotting to determine the binding epitope of DdMab-1. DdMab-1 reacted with the dN670
mutant, but not with the dN680 mutant, indicating that the N-terminus of the DdMab-1 epitope is mainly locatedbetween amino acids 670 and 680 of the protein. Further analysis using point mutants demonstrated that R675A,
R678A, K679A, and K682A mutants were not detected, and V680A was only weakly detected by DdMab-1,
indicating that Arg675, Arg678, Lys679, Val680 and Lys682 are important for binding of DdMab-1 to hDGKδ.

1. Introduction

hetero-oligomerization, and therefore is a putative protein interaction
module [15,16].
DGKδ was previously shown to regulate protein kinase C activity,
and thereby control the degradation of epidermal growth factor receptor
via modulation of ubiquitin-specific protease 8 expression in cultured
human cells [17,18]. Moreover, DGKδ expression and activity levels are
reduced in skeletal muscle tissues of Type 2 diabetic patients [19].
Hence, an anti-DGKδ monoclonal antibody (mAb) is required for specificdetection of DGKδ in human tissues.
In this study, we established a novel anti-human DGKδ (hDGKδ)mAb, DdMab-1, by immunizing mice with recombinant hDGKδ. We also
determined the binding epitope of DdMab-1 using deletion or point
mutants of hDGKδ via Western blot analysis.

Diacylglycerol kinase (DGK) plays a critical role in the regulation ofnumerous cellular functions by catalyzing the phosphorylation of diac­ylglycerol to phosphatidic acid [1,2]. Diacylglycerol activates protein
kinase C, and the DGK terminates the diacylglycerol-mediated signaling
pathway by phosphorylating diacylglycerol [3–7]. Here, the resulting
phosphatidic acid functions as a second messenger which regulates theintracellular Ca2+ level and the mTOR-mediated signaling pathway [8,9].
Ten isozymes of the DGK family have been so far identified in
mammals [2]. DGK family is also grouped into five subtypes based on
their subtype-specific functional domains. DGKδ is one of the DGK
family, and was first cloned from the human testis cDNA library [10].
DGKδ is expressed in several tissues and organs including the stom­ach, testis, bone marrow, and lymph node [11]. DGKδ is a type II DGK
which contains pleckstrin homology (PH) and sterile alpha motif (SAM)
domains at the N- and C-terminus of the protein, respectively. The PH
domain can bind protein kinase C, the βγ-subunits of heterotrimeric G
proteins, and phosphatidylinositol 4,5-bisphosphate [12–14]. On theother hand, the SAM domain has been shown to mediate both homo- and2. Materials and methods
2.1. Plasmid preparation
Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan) encoding
hDGKδ (accession No.NM_152879) plus a C-terminal PA tag (GVAMP­GAEDDVV) [20] was subcloned into the pMAL-c2 expression vector

Abbreviations: DGKδ, Diacylglycerol kinase δ; hDGKδ, human DGKδ; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PH,pleckstrin homology; PVDF, polyvinylidene difluoride; SAM, sterile alpha motif; TBS, Tris-buffered saline.
* Corresponding author. New Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
E-mail addresses: yukinarikato@med.tohoku.ac.jp, yukinarikato@med.tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2020.100808
Received 25 July 2020; Accepted 22 August 2020
2405-5808/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

M. Sano et al.

Biochemistry and Biophysics Reports 24 (2020) 100808(New England Biolabs Inc., Beverly, MA) using the In-Fusion HD Cloning
Kit (Takara Bio, Inc., Shiga, Japan). The PA tag is recognized by ananti-PA tag mAb (NZ-1) [21]. The resulting construct was namedpMAL-c2-hDGKδ-PA. The deletion mutants of hDGKδ DNA were
amplified via polymerase chain reaction, and subcloned into thepMAL-c2 with a PA tag using the In-Fusion HD Cloning Kit. The sub­stitution of hDGKδ amino acids with alanine on dN610 of hDGKδ was
performed using the QuikChange Lightning Site-Directed Mutagenesis
Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). These constructs
were also verified by direct DNA sequencing.
2.2. Production of the recombinant DGKδ protein
Competent E. coli TOP-10 cells (Thermo Fisher Scientific Inc., Wal­
tham, MA, USA) were transformed with the pMAL-c2-hDGKδ-PA
plasmid. The cells were cultured overnight at 37 ◦ C in LB medium(Thermo Fisher Scientific Inc.) containing 100 μg/ml of ampicillin(Sigma-Aldrich Corp., St. Louis, MO). Cell pellets were resuspended inphosphate-buffered saline (PBS) containing 1% Triton X-100 and 50 μg/
ml aprotinin (Sigma-Aldrich Corp.). After sonication, crude extracts
were collected using centrifugation (9000×g, 30 min, 4 ◦ C). The lysates
were passed through a 0.45 μm filter to remove trace amounts ofinsoluble materials. Filtered lysates were then mixed with NZ-1Sepharose (1 ml of bed volume), and incubated at 4 ◦ C for 2 h undergentle agitation. The resulting resin was then transferred to a column,
and washed with 20 ml of Tris-buffered saline (TBS; pH 7.5). The boundprotein was eluted with the PA tag peptide at room temperature in a
stepwise manner (1 ml × 10 washes).
2.3. Hybridoma production

Fig. 1. Epitope mapping of DdMab-1 using deletion mutants of hDGKδ. (A)Schematic illustration of DdMab-1 epitope mapping. Black bars, deletion mu­
tants detected by DdMab-1. hDGKδ, human DGKδ; MBP, maltose-binding pro­tein. (B) Cell lysates of hDGKδ N-terminal deletion mutants were
electrophoresed, and then transferred onto a PVDF membrane. After blocking,the membrane was incubated with 1 μg/ml of DdMab-1 or anti-PA tag antibody(NZ-1).

The Animal Care and Use Committee of Tohoku University approved
all animal experiments. DdMab-1 was produced using the mouse medial
iliac lymph node method. Briefly, three female 8-week old B6D2F1/Slc
mice (Japan SLC Inc., Shizuoka, Japan) were immunized by injecting 33
μg of the pMAL-c2-hDGKδ-PA protein and Freund’s complete adjuvant(Sigma-Aldrich Corp.) into their footpad. Additional immunization with50 μg of the pMAL-c2-hDGKδ-PA protein was performed via the tailbase. The lymphocytes were fused with mouse myeloma Sp2/0-Ag14
cells using polyethylene glycol (PEG). The culture supernatants werescreened using enzyme-linked immunosorbent assay for binding to thepMAL-c2-hDGKδ-PA protein.

immunization with 50 μg of the pMAL-c2-hDGKδ-PA protein was per­formed via the tail base. The lymphocytes were fused with mouse
myeloma Sp2/0-Ag14 cells using PEG. The culture supernatants werescreened using enzyme-linked immunosorbent assay for the binding to
the pMAL-c2-hDGKδ-PA protein. After Western blot screening, weestablished DdMab-1 (mouse IgG2a, kappa), which is useful for Western
blot analysis against hDGKδ (Fig. 1).

2.4. Western blot analyses
Lysates were boiled in sodium dodecyl sulfate sample buffer (Nacalai
Tesque, Inc., Kyoto, Japan). The samples were electrophoresed using
5%–20% polyacrylamide gels under reducing condition (Nacalai Tes­que, Inc.), and transferred onto a polyvinylidene difluoride (PVDF)
membranes (Merck KGaA, Darmstadt, Germany). After blocking with
4% skim milk (Nacalai Tesque, Inc.) for 1 h, the membrane was incu­
bated with DdMab-1 (1 μg/mL or 10 μg/mL) or NZ-1 (1 μg/mL) for 1 h,followed by incubation with HRP-conjugated anti-mouse immunoglob­
ulins (1:2000 dilution; Agilent Technologies, Inc.) or HRP-conjugatedanti-rat IgG (1:10,000 dilution; Sigma-Aldrich Corp.) for 1 h. The
membrane was developed with the ImmunoStar LD Chemiluminescence
Reagent (FUJIFILM Wako Pure Chemical Corporation) using the SayacaImager (DRC Co., Ltd., Tokyo, Japan). All Western blot procedures were
performed at room temperature.

3.2. Epitope mapping of DdMab-1 using deletion mutant of hDGKδ
Because DdMab-1 reacted with an N-terminal deletion mutant(dN570) of hDGKδ, we then produced an additional 14 N-terminal
deletion mutants (dN580, dN590, dN600, dN610, dN620, dN630,
dN640, dN650, dN660, dN670, dN680, dN690, dN700, and dN710), andperformed western blotting to detect the location of the epitope(Fig. 1A). As shown in Fig. 1B, DdMab-1 recognized dN580, dN590,
dN600, dN610, dN620, dN630, dN640, dN650, dN660, and dN670, but
not dN680, dN690, dN700, and dN710 mutants. All of these deletionmutants were detected by the NZ-1 anti-PA tag mAb (Fig. 1B). Hence,
the N-terminus of DdMab-1 epitope was found to be located between
amino acids 670 and 680 of hDGKδ.

3. Results

3.3. Epitope mapping of DdMab-1 using point mutants of hDGKδ

3.1. Establishment of anti-hDGKδ mAbs

We also produced further hDGKδ constructs including 26 alanine
point mutations to identify the critical DdMab-1 epitope (G670A,
V671A, P672A, K673A, G674A, R675A, S676A, Q677A, R678A, K679A,
V680A, S681A, K682A, S683A, P684A, C685A, E686A, K687A, L688A,

Three B6D2F1/Slc mice were immunized by injecting 33 μg of thepMAL-c2-hDGKδ-PA protein into their footpad. Additional
2

M. Sano et al.

Biochemistry and Biophysics Reports 24 (2020) 100808

catalytic and accessory domains (Fig. 2C). In our next study, we will
investigate the utility of this mAb in immunocytochemistry and immu­
nohistochemistry analyses for detection of hDGKδ protein in different
tissues/organs including the stomach, testis, bone marrow, and lymph
node.
Funding
This research was supported in part by AMED [grant numbers:
JP20am0401013, JP20am0101078, JP20ae0101028] (Y.K.).
Transparency document
Transparency document related to this article can be found online athttps://doi.org/10.1016/j.bbrep.2020.100808
References[1] M.K. Topham, R.M. Epand, Mammalian diacylglycerol kinases: molecularinteractions and biological functions of selected isoforms, Biochim. Biophys. Acta
1790 (2009) 416–424, https://doi.org/10.1016/j.bbagen.2009.01.010.
[2] K. Goto, Y. Hozumi, T. Nakano, S.S. Saino, H. Kondo, Cell biology andpathophysiology of the diacylglycerol kinase family: morphological aspects intissues and organs, Int. Rev. Cytol. 264 (2007) 25–63, https://doi.org/10.1016/
s0074-7696(07)64002-9.
[3] M.J. Berridge, R.F. Irvine, Inositol trisphosphate, a novel second messenger incellular signal transduction, Nature 312 (1984) 315–321, https://doi.org/
10.1038/312315a0.
[4] A.C. Newton, Regulation of protein kinase C, Curr. Opin. Cell Biol. 9 (1997)
161–167, https://doi.org/10.1016/s0955-0674(97)80058-0.
[5] D.B. Parekh, W. Ziegler, P.J. Parker, Multiple pathways control protein kinase C
phosphorylation, EMBO J. 19 (2000) 496–503, https://doi.org/10.1093/emboj/
19.4.496.
[6] I. Mérida, J. Arranz-Nicolás, C. Rodríguez-Rodríguez, A. Ávila-Flores,
Diacylglycerol kinase control of protein kinase C, Biochem. J. 476 (2019)1205–1219, https://doi.org/10.1042/bcj20180620.
[7] Y. Nishizuka, The role of protein kinase C in cell surface signal transduction andtumour promotion, Nature 308 (1984) 693–698, https://doi.org/10.1038/
308693a0.
[8] Y. Fang, M. Vilella-Bach, R. Bachmann, A. Flanigan, J. Chen, Phosphatidic acidmediated mitogenic activation of mTOR signaling, Science 294 (2001) 1942–1945,
https://doi.org/10.1126/science.1066015.
[9] D. English, Y. Cui, R.A. Siddiqui, Messenger functions of phosphatidic acid, Chem.
Phys. Lipids 80 (1996) 117–132, https://doi.org/10.1016/0009-3084(96)02549-2.
[10] F. Sakane, S. Imai, M. Kai, I. Wada, H. Kanoh, Molecular cloning of a novel
diacylglycerol kinase isozyme with a pleckstrin homology domain and a C-terminal
tail similar to those of the EPH family of protein-tyrosine kinases, J. Biol. Chem.
271 (1996) 8394–8401, https://doi.org/10.1074/jbc.271.14.8394.
[11] L. Fagerberg, B.M. Hallström, P. Oksvold, C. Kampf, D. Djureinovic, J. Odeberg,
M. Habuka, S. Tahmasebpoor, A. Danielsson, K. Edlund, A. Asplund, E. Sjöstedt,
E. Lundberg, C.A. Szigyarto, M. Skogs, J.O. Takanen, H. Berling, H. Tegel,
J. Mulder, P. Nilsson, J.M. Schwenk, C. Lindskog, F. Danielsson, A. Mardinoglu,
A. Sivertsson, K. von Feilitzen, M. Forsberg, M. Zwahlen, I. Olsson, S. Navani,
M. Huss, J. Nielsen, F. Ponten, M. Uhlén, Analysis of the human tissue-specificexpression by genome-wide integration of transcriptomics and antibody-based
proteomics, Mol. Cell. Proteomics 13 (2014) 397–406, https://doi.org/10.1074/
mcp.M113.035600.
[12] D.S. Wang, G. Shaw, The association of the C-terminal region of beta I sigma II
spectrin to brain membranes is mediated by a PH domain, does not require
membrane proteins, and coincides with a inositol-1,4,5 triphosphate binding site,
Biochem. Biophys. Res. Commun. 217 (1995) 608–615, https://doi.org/10.1006/
bbrc.1995.2818.
[13] K. Touhara, J. Inglese, J.A. Pitcher, G. Shaw, R.J. Lefkowitz, Binding of G protein
beta gamma-subunits to pleckstrin homology domains, J. Biol. Chem. 269 (1994)
10217–10220.
[14] H. Konishi, S. Kuroda, U. Kikkawa, The pleckstrin homology domain of RAC
protein kinase associates with the regulatory domain of protein kinase C zeta,
Biochem. Biophys. Res. Commun. 205 (1994) 1770–1775, https://doi.org/
10.1006/bbrc.1994.2874.
[15] D. Stapleton, I. Balan, T. Pawson, F. Sicheri, The crystal structure of an Eph
receptor SAM domain reveals a mechanism for modular dimerization, Nat. Struct.
Biol. 6 (1999) 44–49, https://doi.org/10.1038/4917.
[16] O. Buzovetsky, C. Tang, K.M. Knecht, J.M. Antonucci, L. Wu, X. Ji, Y. Xiong, The
SAM domain of mouse SAMHD1 is critical for its activation and regulation, Nat.
Commun. 9 (2018) 411, https://doi.org/10.1038/s41467-017-02783-8.
[17] T. Crotty, J. Cai, F. Sakane, A. Taketomi, S.M. Prescott, M.K. Topham,
Diacylglycerol kinase delta regulates protein kinase C and epidermal growth factor
receptor signaling, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 15485–15490,
https://doi.org/10.1073/pnas.0604104103.
[18] J. Cai, T.M. Crotty, E. Reichert, K.L. Carraway 3rd, D.M. Stafforini, M.K. Topham,
Diacylglycerol kinase delta and protein kinase C(alpha) modulate epidermal

Fig. 2. Epitope mapping of DdMab-1 using point mutants of hDGKδ. (A)Cell lysates of point mutants of dN610 were electrophoresed, and then trans­ferred onto PVDF membranes. After blocking, the membranes were incubatedwith 10 μg/ml of DdMab-1 or 1 μg/ml of anti-PA tag antibody (NZ-1). (B)Schematic illustration of DdMab-1 epitope mapping. Underlined amino acids(Arg675, Arg678, Lys679, Val680 and Lys682) are important for binding of
DdMab-1 to hDGKδ. (C) Schematic illustration of the hDGKδ structure. DdMab1 epitope is located in between catalytic and accessory domains. PH, pleckstrin
homology; SAM, sterile alpha motif.

I689A, S690A, K691A, G692A, S693A, L694A, and S695A). All hDGKδpoint mutants were recognized by NZ-1 (Fig. 2A). In contrast, DdMab-1
did not recognize R675A, R678A, K679A, and K682A mutants, and only
weakly reacted with the V680A mutant (Fig. 2A), indicating that
DdMab-1 binds to DGKδ via Arg675, Arg678, Lys679, Val680 andLys682. These results are summarized in Fig. 2B. The identified DdMab1 epitope of is located between the catalytic and accessory domains(Fig. 2C).
4. Discussion
Previously, we established DaMab-2 as an anti-DGKα mAb [22],DgMab-6 as an anti-DGKγ mAb [23], and DzMab-1 as an anti-DGKζ mAb[24] for immunocytochemistry. We further developed DaMab-8 as ananti-DGKα mAb [25] and DhMab-1 [26]/DhMab-4 [27] as anti-DGKηmAbs for immunohistochemistry. We determined their respective
binding epitopes [25–30]. Accordingly, DaMab-2 and DaMab-8 was
found to bind to the Zn-finger and catalytic domains of DGKα, respec­tively [25,28]. DgMab-6 and DzMab-1 were shown to bind to the
N-termini of DGKγ and DGKζ, respectively [29,30]. DhMab-1/DhMab-4
epitope was found to be located near the accessory domain of hDGKη
[26,27]. These epitope analyses revealed that each sensitive and specificmAb for use in immunocytochemistry or immunohistochemistry against
different DGK isotypes included different epitope regions.
Here, we reported a novel anti-hDGKδ mAb, DdMab-1, which isuseful for Western blot analysis (Figs. 1B and 2A). We also identified thebinding epitope of DdMab-1 by western blotting, and found Arg675,
Arg678, Lys679, Val680 and Lys682 to be important for DdMab-1
binding to hDGKδ. The epitope of DdMab-1 is located between
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Biochemistry and Biophysics Reports 24 (2020) 100808[24] T. Nakano, S. Ogasawara, T. Tanaka, Y. Hozumi, M. Sano, Y. Sayama, S. Yamada,
M.K. Kaneko, Y. Kato, K. Goto, DzMab-1: anti-human DGKzeta monoclonal
antibody for immunocytochemistry, Monoclon. Antibodies Immunodiagn.
Immunother. 38 (2019) 179–182.
[25] M. Sano, M.K. Kaneko, H. Suzuki, Y. Kato, Establishment and epitope mapping ofanti-diacylglycerol kinase alpha monoclonal antibody DaMab-8 for
immunohistochemical analyses, Monoclon. Antibodies Immunodiagn.
Immunother. 39 (2020) 1–5, https://doi.org/10.1089/mab.2019.0004.
[26] M. Sano, M.K. Kaneko, Y. Kato, Epitope mapping of DhMab-1: anantidiacylglycerol kinase monoclonal antibody monoclon, Antib. Immunodiagn.
Immunother. (2020), https://doi.org/10.1089/mab.2020.0014.
[27] T. Asano, M. Sano, J. Takei, Y. Sayama, M.K. Kaneko, Y. Kato, Epitope mapping of
the anti-diacylglycerol kinase monoclonal antibody DhMab-4 forimmunohistochemical analysis, Monoclon. Antibodies Immunodiagn. Immunother.
(2020), https://doi.org/10.1089/mab.2020.0012.
[28] M. Sano, M.K. Kaneko, Y. Kato, Epitope mapping of antidiacylglycerol kinase alphamonoclonal antibody DaMab-2, Monoclon. Antibodies Immunodiagn.
Immunother. 38 (2019) 8–11, https://doi.org/10.1089/mab.2018.0047.
[29] M. Sano, M.K. Kaneko, Y. Kato, Epitope mapping of antihuman diacylglycerol
kinase gamma monoclonal antibody DgMab-6, Monoclon. Antibodies
Immunodiagn. Immunother. 38 (2019) 120–123, https://doi.org/10.1089/
mab.2019.0011.
[30] M. Sano, M.K. Kaneko, Y. Kato, Epitope mapping of anti-diacylglycerol kinase zeta
monoclonal antibody DzMab-1 for immunohistochemical analyses, Monoclon.
Antibodies Immunodiagn. Immunother. 38 (2019) 175–178, https://doi.org/
10.1089/mab.2019.0020.

growth factor receptor abundance and degradation through ubiquitin-specificprotease 8, J. Biol. Chem. 285 (2010) 6952–6959, https://doi.org/10.1074/jbc.
M109.055731.
[19] A.V. Chibalin, Y. Leng, E. Vieira, A. Krook, M. Björnholm, Y.C. Long, O. Kotova,
Z. Zhong, F. Sakane, T. Steiler, C. Nylén, J. Wang, M. Laakso, M.K. Topham,
M. Gilbert, H. Wallberg-Henriksson, J.R. Zierath, Downregulation of diacylglycerol
kinase delta contributes to hyperglycemia-induced insulin resistance, Cell 132(2008) 375–386, https://doi.org/10.1016/j.cell.2007.12.035.
[20] Y. Fujii, M. Kaneko, M. Neyazaki, T. Nogi, Y. Kato, J. Takagi, PA tag: a versatile
protein tagging system using a super high affinity antibody against adodecapeptide derived from human podoplanin, Protein Expr. Purif. 95 (2014)240–247, https://doi.org/10.1016/j.pep.2014.01.009.
[21] Y. Kato, M.K. Kaneko, A. Kuno, N. Uchiyama, K. Amano, Y. Chiba, Y. Hasegawa,J. Hirabayashi, H. Narimatsu, K. Mishima, M. Osawa, Inhibition of tumor cellinduced platelet aggregation using a novel anti-podoplanin antibody reacting with
its platelet-aggregation-stimulating domain, Biochem. Biophys. Res. Commun. 349(2006) 1301–1307, https://doi.org/10.1016/j.bbrc.2006.08.171.
[22] T. Nakano, S. Ogasawara, T. Tanaka, Y. Hozumi, S. Mizuno, E. Satoh, F. Sakane,
N. Okada, A. Taketomi, R. Honma, T. Nakamura, N. Saidoh, M. Yanaka, S. Itai,
S. Handa, Y.W. Chang, S. Yamada, M.K. Kaneko, Y. Kato, K. Goto, DaMab-2: antihuman DGKalpha monoclonal antibody for immunocytochemistry, Monoclon.
Antibodies Immunodiagn. Immunother. 36 (2017) 181–184, https://doi.org/
10.1089/mab.2017.0023.
[23] T. Nakano, S. Ogasawara, T. Tanaka, Y. Hozumi, A. Yamaki, F. Sakane, Y. Shirai,
T. Nakamura, M. Yanaka, S. Yamada, M.K. Kaneko, Y. Kato, K. Goto, DgMab-6:
antihuman DGKgamma monoclonal antibody for immunocytochemistry,
Monoclon. Antibodies Immunodiagn. Immunother. 37 (2018) 229–232, https://doi.org/10.1089/mab.2018.0026.

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