Biochemical and Biophysical Research Communications 459 (2015) 636e642

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

A novel monoclonal antibody SMab-2 recognizes endogenous
IDH2-R172S of chondrosarcoma
Xing Liu a, b, Satoshi Ogasawara a, Mika K. Kaneko a, Hiroharu Oki a, b, Yasukazu Hozumi c,
Kaoru Goto c, Michiaki Takagi b, Yukinari Kato a, *abcDepartment of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan
Department of Anatomy and Cell Biology, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 25 February 2015
Available online 6 March 2015

Isocitrate dehydrogenase 2 (IDH2) mutations have been reported in gliomas, osteosarcomas, cartilaginous tumors, giant cell tumors of bone, and acute myeloid leukemias. Although IDH2 catalyzes theoxidative carboxylation of isocitrate to a-ketoglutarate (a-KG) in mitochondria, mutated IDH2 proteins
possess the ability to change a-KG into the oncometabolite R(-)-2-hydroxyglutarate (2-HG). To date,
several monoclonal antibodies (mAbs) speciﬁc for IDH2 mutations have been established, such as KMab1 against IDH2-R172K, MMab-1 against IDH2-R172M, and WMab-1 against IDH2-R172W. Although amulti-speciﬁc mAb MsMab-1 reacted with IDH2-R172G and IDH2-R172S, a mono-speciﬁc mAb against
IDH2-R172S has not been established. In this study, we established a novel mAb SMab-2, which recognizes IDH2-R172S but not with wild type IDH2 in ELISA. Although SMab-2 reacted with both IDH1R132S and IDH2-R172S expressed in Escherichia coli, it reacted with only IDH2-R172S expressed in U-2
OS osteosarcoma cells. Furthermore, SMab-2 recognized endogenous IDH2-R172S protein expressed in
SW1353 chondrosarcoma cells in Western blot and immunocytochemical analyses. SMab-2 is expected
to be useful for diagnosis of IDH2-R172S-bearing tumors.
© 2015 Elsevier Inc. All rights reserved.

Keywords:
IDH2
IDH2 mutation
R172S
Monoclonal antibody
SMab-2

1. Introduction
Somatic mutations of isocitrate dehydrogenase 2 (IDH2) wereﬁrst found in gliomas [1]. IDH2 convert a-ketoglutarate (a-KG) tooncometabolite R(-)-2-hydroxyglutarate (2-HG), although IDH2
catalyzes the oxidative carboxylation of isocitrate to a-ketoglutarate in mitochondria [2]. IDH2 mutations, which are speciﬁc to asingle codon in arginine 172 residue (R172), were also reported inosteosarcomas [3], giant cell tumors of bone [4], and in cartilaginous tumors [5e7]. In contrast, IDH2 mutations of acute myeloid
leukemias [8,9] were discovered in not only R172 but also arginine
140 residue (R140), which is found more frequently than R172 [10].
Equivocal microscopic diagnosis often occurs from several factors

Abbreviations: IDH1/2, isocitrate dehydrogenase 1/2; mAb, monoclonal
antibody.
* Corresponding author. Fax: þ81 22 717 8207.
E-mail addresses: yukinari-k@bea.hi-ho.ne.jp, yukinarikato@med.tohoku.ac.jp(Y. Kato).
http://dx.doi.org/10.1016/j.bbrc.2015.02.162
0006-291X/© 2015 Elsevier Inc. All rights reserved.

such as small sample size, sampling site, or sample quality. IDH
status provides clinically important information in those cases
especially in gliomas, because IDH status is correlated with glioma
patient prognosis [1]. There are several methods for testing IDH
status, which targets DNA sequence, protein, and 2-HG [11]. DNA
direct sequencing (Sanger method) and immunohistochemistry are
two conventional methods, which are applied for daily diagnostic
practice. Recently, several special methods such as pyrosequencing[12], melting curve analysis [13], and magnetic resonance spectroscopy (MRS) [14] were developed; however, those methods
need special equipment [11]. Although Sanger method detects all
types of mutations, at least 20% of the mutant allele is required for
detection of IDH mutations [12]. In contrast, immunohistochemistry detects only one mutation-bearing tumor cell; however, many
types of monoclonal antibodies (mAbs) against IDH mutations
should be developed. To date, we established several mono-speciﬁc
anti-mutated IDH2 mAbs [15]: IDH2-R172K-speciﬁc KMab-1 [16],IDH2-R172M-speciﬁc MMab-1 [16], and IDH2-R172W-speciﬁc
WMab-1 [17]. We also established two multi-speciﬁc IDH mAbs:
MsMab-1 [18] and MsMab-2 [19]. In this study, we report a novel

X. Liu et al. / Biochemical and Biophysical Research Communications 459 (2015) 636e642

mAb SMab-2, which recognizes IDH2-R172S but not with wild type
IDH.
2. Materials and methods
2.1. Cell lines
U-2 OS osteosarcoma cell line, SW1353 chondrosarcoma cellline, HeLa cervical adenocarcinoma cell lines, and P3X63Ag8U.1(P3U1) mouse myeloma cell line were obtained from the American
Type Culture Collection (ATCC, Manassas, VA), and were cultured at
37  C in a humidiﬁed atmosphere of 5% CO2 and 95% air. U-2 OS,
SW1353, and HeLa were cultured in Dulbecco's modiﬁed Eagle
medium (DMEM; Nacalai Tesque Inc., Kyoto, Japan) and P3U1 wascultured in Roswell Park Memorial Institute (RPMI) 1640 medium(Nacalai Tesque Inc.), including 2 mM L-glutamine (Nacalai TesqueInc.) supplemented with 10% heat-inactivated fetal bovine serum(FBS; Life Technologies Inc., Carlsbad, CA).
2.2. Hybridoma production
BALB/c mice were immunized by intraperitoneal (i.p.) injectionsof 125 mg of synthetic peptides of CGGVKPITIGSHAHGDQYKA (IDH2R172S) or CMWKSPNGTIQNILGGTVFR (IDH2-R140Q), conjugatedwith keyhole limpet hemocyanin (KLH) together with Imject Alum
(Thermo Fisher Scientiﬁc Inc., Waltham, MA). One week later, secondary i.p. immunization of 62.5 mg of synthetic peptides was performed. After several additional immunizations of 62.5 mg ofsynthetic peptides, a booster injection was given i.p. 2 days before
spleen cells were harvested. The spleen cells were fused with P3U1
cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in RPMI1640 medium with hypoxanthine,aminopterin, and thymidine (HAT) selection medium supplement(Life Technologies Corp.). The culture supernatants were screenedusing enzyme-linked immunosorbent assay (ELISA).
2.3. Enzyme-linked immunosorbent assay (ELISA)

637

Ultrasonics Corp., Danbury, CT), the crude extracts were collected bycentrifugation (9000  g, 30 min, 4  C). The supernatants were
loaded onto amylose resin (New England Biolabs Inc.). The loaded
resins were washed extensively with column buffer (20 mM
TriseHCl, pH 7.4, 200 mM NaCl, 1 mM EDTA). The fusion proteins
were eluted by column buffer with 10 mM maltose. Then U-2 OS cells
were transfected with appropriate amounts of plasmids,
pcDNA3.1VH/IDH1-WT, pcDNA3.1VH/IDH1-R132H, pcDNA3.1VH/
IDH1-R132C, pcDNA3.1VH/IDH1-R132S, pcDNA3.1VH/IDH1-R132G,
pcDNA3.1VH/IDH1-R132L, pcDNA3-PAcH/IDH2-WT, pcDNA3-PAcH/
IDH2-R172K, pcDNA3-PAcH/IDH2-R172M, pcDNA3-PAcH/IDH2R172W, pcDNA3-PAcH/IDH2-R172S, pcDNA3-PAcH/IDH2-R172G using Lipofectamine LTX (Life Technologies Inc.) according to the
manufacturer's instructions [3]. The expression level of IDH1/2 was
conﬁrmed using Western blot analyses.
2.5. Western blot analyses
Cultured cell pellets were lysed with 1% RIPA buffer (Thermo
Fisher Scientiﬁc Inc.) for 15 min on ice. The lysate supernatants
were centrifuged for 15 min at 15,000 rpm to remove cellulardebris. Cell lysates were prepared for Western blot analyses byboiling in SDS sample buffer (Nacalai Tesque, Inc.). They were
electrophoresed on 5e20% polyacrylamide gels (Wako Pure
Chemical Industries Ltd.). The separated proteins were transferred
to a PVDF membrane (EMD Millipore Corp., Billerica, MA). After
blocking with 4% skim milk in PBS with 0.05% Tween 20 for 15 min,the membrane was incubated with 10 mg/ml of SMab-2, 5 mg/ml of
RqMab-3, or 1 mg/ml of SMab-1 [20], RcMab-1 [19], 5F11 (antiIDH2; SigmaeAldrich Corp.), 1H6 (anti-V5 tag; Medical & Biological
Laboratories Co. Ltd., Nagoya, Japan), NZ-1 (anti-PA tag) [21,22], andAC-15 (anti-b-actin; SigmaeAldrich Corp.) for 60 min. Then themembrane was incubated with peroxidase-conjugated secondary
antibodies (1:1000 diluted; Dako) for 30 min, and developed with
ImmunoStar LD Chemiluminescence Reagent (Wako Pure Chemical
Industries, Ltd., Osaka, Japan) using Sayaca-Imager (DRC Co. Ltd.,Tokyo, Japan).

Synthetic peptides or recombinant proteins were immobilizedon Nunc Maxisorp 96-well immunoplates (Thermo Fisher ScientiﬁcInc.) at 1 mg/ml for 30 min. Synthetic peptides are as follows:
GGTKPITIGRHAHGDQYKA (IDH2-WT), GGTKPITIGSHAHGDQYKA(IDH2-R172S). After blocking with SuperBlock T20 (PBS) Blocking
Buffer (Thermo Fisher Scientiﬁc Inc.), the plates were incubatedwith culture supernatant or puriﬁed mAbs (1 mg/ml) with subsequent 1:1000 diluted peroxidase-conjugated anti-mouse IgG(Dako, Glostrup, Denmark). The enzymatic reaction was conductedwith 1-Step Ultra TMB-ELISA (Thermo Fisher Scientiﬁc Inc.). The
optical density was measured at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories Inc., Philadelphia, PA). These reactions
were performed with a volume of 50 ml at 37  C.

Genomic DNA was extracted from SW1353 chondrosarcoma cellline according to the manufacturer's instructions (Takara Bio Inc.,
Shiga, Japan). PCR primers for the genomic region corresponding to
IDH2 exon 4, which encodes codon R172, and the ﬂanking intronic
sequences were the following: human IDH2 sense (50 -CAAGCTGAAGAAGATGTGGAA-30 ) and human IDH2 antisense (50 -CAGAGACAAGAGGATGGCTA-30 ). The PCR conditions were 98  C for 2 min (1cycle), followed by 40 cycles of 98  C for 10 s, 60  C for 15 s, 68  C for
30 s, and extension at 68  C for 10 min with MightyAmp DNA polymerase (Takara Bio Inc.). Cycle sequencing was conducted using
the sequencing primer for IDH2 (50 -AGCCCATCATCTGCAAAAAC-30 ).

2.4. Protein expression using bacteria cells and mammalian cells

2.7. Droplet digital PCR analysis

Competent Escherichia coli TOP-10 cells (Life Technologies Inc.)
were transformed with appropriate amounts of plasmids, pMALIDH1-WT, pMAL-IDH1-R132H, pMAL-IDH1-R132C, pMAL-IDH1R132S, pMAL-IDH1-R132G, pMAL-IDH1-R132L, pMAL-IDH2-WT,
pMAL-IDH2-R172K,
pMAL-IDH2-R172M,
pMAL-IDH2-R172W,
pMAL-IDH2-R172S, and pMAL-IDH2-R140Q [18]. Then, they were
cultured overnight at 37  C in LB medium (Life Technologies Inc.)
containing 100 mg/ml ampicillin (Sigma-Aldrich Corp., St. Louis, MO).
Cell pellets were resuspended in phosphate buffered saline (PBS)
with 1% Triton X-100 with 50 mg/ml aprotinin (SigmaeAldrich Corp.).
After sonication using Branson Advanced Soniﬁer (Branson

The droplet digital PCR analysis was performed as described
previously [23]. Brieﬂy, the TaqMan PCR reaction mixture wasassembled from a 2  ddPCR Supermix (Bio-Rad Laboratories Inc.),
20  primer and probe (wild type), 20  primer and probe (IDH2R172S), and template in a ﬁnal volume of 20 ml. Each assembled
ddPCR reaction mixture was then loaded into the sample well of aneight-channel disposable droplet generator cartridge (Bio-Rad
Laboratories Inc.). A volume of 70 ml of droplet generation oil (BioRad Laboratories Inc.) was loaded into the oil well for each channel.
The cartridge was placed into the droplet generator (Bio-Rad Laboratories Inc.). The cartridge was removed from the droplet

2.6. Direct DNA sequencing of IDH2 mutation

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X. Liu et al. / Biochemical and Biophysical Research Communications 459 (2015) 636e642

generator, where the droplets were then manually transferred witha multichannel pipet to a 96-well PCR plate. The plate was heatsealed with a foil seal and then placed on a conventional thermal
cycler and ampliﬁed to the end-point (39 cycles). After PCR, the 96well PCR plate was loaded on the droplet reader (Bio-Rad Laboratories Inc.). Analysis of the ddPCR data was performed with
QuantaSoft analysis software (Bio-Rad Laboratories Inc.) that
accompanied the droplet reader.

(Life Technologies Inc.) to stain the cell nuclei. They were examinedusing confocal laser-scanning microscopy (LSM700; Carl Zeiss Inc.,
Jena, Germany). Then ﬂuorescent images were processed using
image-processing software (Adobe Photoshop; Adobe Systems
Inc.).
3. Results

2.8. Immunocytochemical analyses

3.1. Production of a mutated IDH2-R172S-speciﬁc monoclonal
antibody

Cultured cells were ﬁxed with 4% paraformaldehyde (Wako Pure
Chemical Industries Ltd.) in 0.1 M phosphate buffer (pH 7.4) for
20 min at room temperature, and permeabilized with 0.1% Triton X100 in PBS for 15 min. Cells were then treated with 10% normal goat
serum in PBS (NGS/PBS) to block nonspeciﬁc binding sites, andwere incubated with 30 mg/ml of SMab-2 or control (PBS) containing 0.1% Triton X-100 overnight at 4  C in a moist chamber. They
were incubated with goat anti-mouse IgG-Alexa 488 (dilution
1:200; Life Technologies Corp.) in PBS containing 0.1% Triton X-100
for 1 h at room temperature. Cells were also treated with TO-PRO-3

We immunized mice with synthetic peptides of IDH2-R172S,
and screened IDH2-R172S-reactive/IDH2-wild type (WT)-nonreactive mAbs using ELISA. After limiting dilution, clone SMab-2(mouse IgG1, kappa) against IDH2-R172S was established. SMab-2
also reacted with MBP-fused IDH2-R172S protein, not with MBPfused IDH2-WT protein using ELISA. We further immunized micewith synthetic peptides of IDH2-R140Q, and screened mAbs
reacting with both IDH1-WT and IDH2-WT using ELISA. After
limiting dilution, clone RqMab-3 (mouse IgG1, kappa) against IDH1/
2-WT was established.

Fig. 1. Western-blot analyses by anti-IDH1/2 mAbs against MBP-IDH1/2 proteins. Puriﬁed proteins (50 ng/lane) of E. coli-expressing IDH1 wild type (WT; lane 1), IDH1 mutants(lane 2, IDH1-R132H; lane 3, IDH1-R132C; lane 4, IDH1-R132S; lane 5, IDH1-R132G; lane 6, IDH1-R132L), IDH2-WT (lane 7), IDH2 mutants (lane 8, IDH2-R172K; lane 9, IDH2R172M; lane 10, IDH2-R172W; lane 11, IDH2-R172S; lane 12, IDH2-R140Q) were electrophoresed under reducing condition using 5e20% gels, and were Western-blotted with
SMab-2, SMab-1 (anti-IDH1-R132S), RcMab-1 (anti-IDH1), 5F11 (anti-IDH2), RqMab-3 (anti-IDH1/2), and TMab-2 (anti-MBP).

X. Liu et al. / Biochemical and Biophysical Research Communications 459 (2015) 636e642

3.2. Speciﬁcity of SMab-2 against IDH in western blot analyses
To determine the speciﬁcity of SMab-2, IDH1/2-WT and IDH1/2
mutants (MBP-IDH1-R132H, MBP-IDH1-R132C, MBP-IDH1-R132S,
MBP-IDH1-R132G, MBP-IDH1-R132L, MBP-IDH2-R172K, MBPIDH2-R172M, MBP-IDH2-R172W, MBP-IDH2-R172S, MBP-IDH2R140Q) were Western-blotted using several anti-IDH1/2 mAbs. As
shown in Fig. 1, RcMab-1 (anti-IDH1) recognized IDH1-WT and all
IDH1 mutations. 5F11 (anti-IDH2) reacted with IDH2-WT and all
IDH2 mutations. RqMab-3 (anti-IDH1/2) detected IDH1/2-WT and
all IDH1/2 mutations. TMab-2 (anti-MBP tag) detected all IDH1/2
proteins. In contrast, SMab-2 strongly recognized MBP-IDH2R172S, and weakly reacted with MBP-IDH1-R132S. SMab-1 reacted with only MBP-IDH1-R132S, not with MBP-IDH2-R172S.

639

We next performed Western-blot analyses against IDH1/2 mutations, which are expressed in mammalian cells. IDH1/2-WTs andIDH1/2 mutants (IDH1-R132H, IDH1-R132C, IDH1-R132S, IDH1R132G, IDH1-R132L, IDH2-R172K, IDH2-R172M, IDH2-R172W,
IDH2-R172S, IDH2-R172G) were transfected in U-2 OS, andWestern-blotted with anti-IDH mAbs. As shown in Fig. 2, RcMab-1
detected both exogenous and endogenous IDH1 proteins. 5F11
reacted with both exogenous and endogenous IDH2 proteins.
RqMab-3 (anti-IDH1/2) recognized both exogenous and endogenous IDH1/2 proteins. Anti-tag mAbs, 1H6 (anti-V5 tag) and NZ-1
(anti-PA tag) reacted with exogenous IDH1 and IDH2 proteins,respectively. SMab-2 strongly recognized IDH2-R172S, but notreacted with IDH1/2-WTs. In contrast, SMab-1 recognized IDH1R132S, not with other mutations. Interestingly, SMab-2 detected

Fig. 2. Western-blot analyses by anti-IDH1/2 mAbs against mutated IDH1/2-expressing U-2 OS. Total cell lysate (10 mg/lane) from U-2 OS cells expressing IDH1 wild type (WT, lane
1) and IDH1 mutants (lane 2, IDH1-R132H; lane 3, IDH1-R132C; lane 4, IDH1-R132S; lane 5, IDH1-R132G; lane 6, IDH1-R132L), IDH2-WT (lane 7), IDH2 mutants (lane 8, IDH2R172K; lane 9, IDH2-R172M; lane 10, IDH2-R172W; lane 11, IDH2-R172S; lane 12, IDH2-R172G) were electrophoresed under reducing condition using 5e20% gels, and wereWestern-blotted with SMab-2, SMab-1 (anti-IDH1-R132S), RcMab-1 (anti-IDH1), 5F11 (anti-IDH2), RqMab-3 (anti-IDH1/2), 1H6 (anti-V5 tag), NZ-1 (anti-PA tag), and AC-15 (anti-bactin). Arrows: endogenous IDH1/2.

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X. Liu et al. / Biochemical and Biophysical Research Communications 459 (2015) 636e642

with only IDH2-R172S in mammalian cells in the different mannerwith E. coli proteins, indicating that SMab-2 is mono-speciﬁc
against IDH2-R172S in cancers.
3.3. SMab-2 reacts with endogenous IDH2-R172S expressed in achondrosarcoma cell line
A chondrosarcoma cell line SW1353 is harboring endogenous
IDH2-R172S (Fig. 3A). The IDH2-R172S mutation of SW1353 was
conﬁrmed using droplet digital PCR analysis (Fig. 3B). In
Western-blot analysis, SMab-2 detected both exogenous IDH2R172S in U-2 OS and endogenous IDH2-R172S in SW1353(Fig. 3C). An anti-IDH2 mAb, 5F11 reacted both wild type andmutated IDH2 proteins. An anti-PA tag mAb, NZ-1 detected only
exogenous IDH2 proteins. These results demonstrate that SMab-2
recognizes not only exogenous IDH2-R172S but also endogenous
one, indicating that SMab-2 is useful for detecting IDH2-R172S intumors.
We next performed immunocytochemical analyses using SMab2 against SW1353 chondrosarcoma cell line. As shown in Fig. 4B,
SMab-2 clearly stained SW1353 cells. In contrast, SMab-2 did notstain HeLa cells, which do not possess IDH2-R172S mutation(Fig. 4B), indicating that SMab-2 is also speciﬁc against IDH2-R172S
in immunocytochemical analyses.

4. Discussion
We previously established several mono-speciﬁc anti-mutated
IDH2 mAbs: IDH2-R172K-speciﬁc KMab-1, IDH2-R172M-speciﬁc
MMab-1, and IDH2-R172W-speciﬁc WMab-1 [15]. We also established two multi-speciﬁc IDH1/2 mAb: MsMab-1 and MsMab-2[15]. Those mAbs have been used as a useful diagnostic tool for
detecting IDH2 mutations in several tumors. However, anti-IDH2R172S-speciﬁc mAbs have not been developed. In this study, wedeveloped clone SMab-2 (mouse IgG1, kappa) against IDH2-R172S.
IDH2-R172S has been discovered in several tumors such as gliomas,
osteosarcomas, and chondrosarcomas; therefore, SMab-2 is advantageous for detecting IDH2-R172S in IDH2-R172S-bearing
tumors.
Although SMab-2 strongly recognized MBP-IDH2-R172S, andweakly reacted with MBP-IDH1-R132S, it detected with only IDH2R172S in mammalian cells. Indeed, 19 amino acids of IDH2-R172S
peptide (GGTKPITIGSHAHGDQYKA) show 78.9% homology with theequivalent portion of IDH1-R132S (GGVKPIIIGSHAYGDQYRA);
therefore, the weak reaction of SMab-2 to MBP-IDH1-R132S isthough to be the cross-reaction. The same cross-reaction of SMab-1
against IDH1-R132S was observed in ELISA (data not shown). A
clone RqMab-3, which was produced by immunizing synthetic
peptide (NGTIQNILGG), detected both IDH1 and IDH2 (Figs. 1 andFig. 3. Endogenous IDH2-R172S mutation in SW1353 cells was detected using three methods. (A) Direct sequencing. Both AGG (WT) and AGT (R172S) were detected. (B) Digital PCR
analysis. Both WT and R172S were detected in SW1353. (C) Western blot analysis. Total cell lysate from U-2 OS cells expressing IDH2-WT (lane 1) and IDH2-R172S (lane 2) or
SW1353 (lane 3) were electrophoresed under reducing condition using 5e20% gels, and were Western-blotted with SMab-2, NZ-1 (anti-PA tag), 5F11 (anti-IDH2), and AC-15 (anti-bactin).

X. Liu et al. / Biochemical and Biophysical Research Communications 459 (2015) 636e642

641

Fig. 4. Immunocytochemical analyses using SMab-2. SW1353 cells (A) and HeLa cells (B) were stained by 30 mg/ml of SMab-2 or control (PBS), followed by anti-mouse IgG-Alexa
488 antibody (green). Cells were also treated with TO-PRO-3 to stain the cell nuclei (blue). Scale bar: 20 mm. (For interpretation of the references to color in this ﬁgure legend, thereader is referred to the web version of this article.)2), and a previously established multi-speciﬁc mAb, clone MsMab-1
also detected both IDH1-R132S and IDH2-R172S [18]. An antiIDH1-R132H mAb, clone H09 cross-reacts with IDH1-R132L [15],indicating that the cross-reaction has been often observed in antiIDH1/2 mAbs. A chondrosarcoma cell line SW1353 was reported topossess endogenous IDH2-R172S [6]. Because anti-IDH2-R172S
mAbs have not been established, the detection of IDH2-R172S was
performed using Sanger sequencing, next generation sequencing,pyrosequencing, or digital PCR. In this study, we revealed that
SMab-2 detected exogenous IDH2-R172S in SW1353 in Westernblot (Fig. 3C) and immunocytochemical analyses (Fig. 4A), indicating that SMab-2 is effective for a diagnostic tool of IDH2-R172Sharboring tumors.
Taken together, SMab-2 is expected to be useful for diagnosis of
IDH2-R172S-bearing tumors. The combination of SMab-2 withpreviously established anti-mutated IDH1/2 mAbs might lead tohigh-sensitive detection of IDH1/2 mutation in clinical diagnosis.
The concentration of SMab-2 needs to be more than 30 mg/ml in
immunocytochemical analyses, showing that SMab-2 is not sosensitive for detecting IDH2-R172S in immunocytochemical analyses. Indeed, SMab-2 could not detect IDH2-R172S protein in
immunohistochemical analyses using parafﬁn-embedded tissues.

We should further produce much more sensitive mAbs against
endogenous IDH2-R172S protein, which are clinically useful inimmunohistochemical analyses.
Author disclosure statement
The authors have no ﬁnancial interest to disclose.
Conﬂict of interest
None.
Acknowledgments
We thank Takuro Nakamura, Kanae Yoshida, and Noriko Saidoh
for their excellent technical assistance. This work was supported inpart by the Platform for Drug Discovery, Informatics, and Structural
Life Science (PDIS) from Japan Agency for Medical Research anddevelopment, AMED (Y.K.); by the Basic Science and Platform
Technology Program for Innovative Biological Medicine from AMED
(Y.K.); by the Regional Innovation Strategy Support Program from
the Ministry of Education, Culture, Sports, Science and Technology

642

X. Liu et al. / Biochemical and Biophysical Research Communications 459 (2015) 636e642(MEXT) of Japan (Y.K.); by Health Labour Sciences Research Grant
from AMED (Y.K.); and by a Grant-in-Aid for Scientiﬁc Research (C)(M.K.K., Y.K.) from MEXT of Japan.
Transparency document
Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.bbrc.2015.02.162.
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