Biochemical and Biophysical Research Communications 432 (2013) 40–45

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

Establishment of novel monoclonal antibodies KMab-1 and MMab-1 speciﬁc
for IDH2 mutations
Mika Kato Kaneko a,b,1, Shunpei Morita b,1, Yuta Tsujimoto b,1, Ryo Yanagiya b, Kana Nasu b, Hiroko Sasaki b,
Yasukazu Hozumi c, Kaoru Goto c, Atsushi Natsume d, Mika Watanabe e, Toshihiro Kumabe f,
Shingo Takano g, Yukinari Kato a,b,⇑a

Regional Innovation Strategy Support Program, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
Molecular Tumor Marker Research Team, Global COE Program, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan
Department of Anatomy and Cell Biology, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japand
Department of Neurosurgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japane
Department of Pathology and Histotechnology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japanf
Department of Neurosurgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japang
Department of Neurosurgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japanbca r t i c l e

i n f oArticle history:
Received 23 January 2013
Available online 31 January 2013
Keywords:
IDH2 mutation
R172K
R172M
Monoclonal antibody

a b s t r a c t
Isocitrate dehydrogenase 1/2 (IDH1/2) mutations have been detected in gliomas, cartilaginous tumors,and leukemias. IDH1/2 mutations are early and frequent genetic alterations, are speciﬁc to a single codon
in the conserved and functionally important Arginine 132 (R132) in IDH1 and Arginine 172 (R172) in
IDH2. We previously established several monoclonal antibodies (mAbs), which are speciﬁc for IDH1
mutations: clones IMab-1 or HMab-1 against IDH1-R132H or clone SMab-1 against IDH1-R132S. However, speciﬁc mAbs against IDH2 mutations have not been reported. To establish IDH2-mutation-speciﬁc
mAbs, we immunized mice or rats with each mutation-containing IDH2 peptides including IDH2-R172K
and IDH2-R172M. After cell fusion, IDH2 mutation-speciﬁc mAbs were screened in Enzyme-Linked
Immunosorbent Assay (ELISA). Established mAbs KMab-1 and MMab-1 reacted with the IDH2-R172K
and IDH2-R172M peptides, respectively, but not with IDH2-wild type (WT) in ELISA. Western-blot analysis also showed that KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M recombinant
proteins, respectively, not with IDH2-WT or other IDH2 mutants, indicating that KMab-1 and MMab-1
are IDH2-mutation-speciﬁc. Furthermore, MMab-1 speciﬁcally stained the IDH2-R172M-expressing cells
in immunocytochemistry, but did not stain IDH2-WT and other IDH2-mutation-containing cells. In
immunohistochemical analysis, MMab-1 speciﬁcally stained IDH2-R172M-expressing glioma. This is
the ﬁrst report to establish anti-IDH2-mutation-speciﬁc mAbs, which could be useful in diagnosis ofmutation-bearing tumors.
Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction
Isocitrate dehydrogenase 1/2 (IDH1/2) mutations have been detected in some gliomas [1–4], cartilaginous tumors [5–7], andacute myeloid leukemias (AML) [8,9]. In astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary glioblastomas,
IDH1/2 mutations have been identiﬁed as early and frequent ge-

Abbreviations: IDH2, isocitrate dehydrogenase 2; mAb, monoclonal antibody.

⇑ Corresponding author at: Regional Innovation Strategy Support Program,Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai,
Miyagi 980-8575, Japan. Fax: +81 23 628 5295.
E-mail addresses: yukinari-k@bea.hi-ho.ne.jp, yukinarikato@med.tohoku.ac.jp(Y. Kato).
1
These authors contributed equally to this work.
0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2013.01.088

netic alterations (IDH1: 50–93%; IDH2: 3–5%), and might be theinitiating event in these glioma subtypes [1–4]. In contrast, primary glioblastomas rarely contain IDH1/2 mutations. The IDH
mutations are remarkably speciﬁc to a single codon in the conserved and functionally important Arginine 132 residue (R132) in
IDH1 and Arginine 172 residue (R172) in IDH2 in gliomas. Of interest, IDH1/2 mutations were observed subsequently in 19–22% of
AML, because the IDH mutations were also discovered in Arginine
140 residue (R140) of IDH2, which are more frequent than R172
[8,9]. IDH1/2 mutations were found to result in the ability of the
enzyme to catalyze the reduced NADP-dependent reduction of aketoglutarate to onco-metabolite R()-2-hydroxyglutarate (2-HG)[10]. Excess accumulation of 2-HG leads to an elevated risk ofmalignant brain tumors in patients with inborn errors of 2-HG
metabolism.

M.K. Kaneko et al. / Biochemical and Biophysical Research Communications 432 (2013) 40–45

To date, four monoclonal antibodies (mAbs) against IDH1 mutations have been reported [11–15]. Of those antibodies, we established IMab-1 and HMab-1, which are speciﬁc for IDH1-R132H,
the most frequent IDH1 mutations (>90%) [14] and SMab-1: antiIDH1-R132S-speciﬁc mAb [13]. IMab-1 and HMab-1 reacted with
the IDH1-R132H peptide, but not with the wild type IDH1 (IDHWT) peptide in ELISA. In Western-blot analysis, IMab-1 andHMab-1 reacted only with the IDH1-R132H protein, not the
IDH1-WT protein or other IDH1 mutants. SMab-1 reacted with
IDH1-R132S speciﬁcally in the same way [13]. In our recent study,
164 cases of glioma were evaluated immunohistochemically for
IDH1 mutations (R132H and R132S) using anti-IDH1 mAbs:
HMab-1 and SMab-1 [11]. IDH1 mutation was detected, respectively, in 9.7%, 63.6%, 51.7%, and 77.8% of primary grade IV, secondary grade IV, grade III, and grade II gliomas. IDH1 mutation as
evaluated using immunohistochemistry might be of greater prognostic signiﬁcance than histological grading alone in grade III and
grade IV gliomas.
Although HMab-1 and SMab-1 are able to ﬁnd more than 90% of
IDH1 mutations of gliomas in immunohistochemistry, IDH2 mutations, which are detected in 3–5% of gliomas, have been missed
unfortunately in immunohistochemistry. Therefore, novel antibodies that recognize IDH2-mutations should be developed to cover allof IDH1/2 mutations in immunohistochemistry. Here, we newly report anti-IDH2-mutation-speciﬁc monoclonal antibodies (KMab-1
and MMab-1), which are expected to be extremely useful for diagnosis of mutation-bearing gliomas.
2. Materials and methods
2.1. Cell lines and tissues
P3X63Ag8U.1 (P3U1), Sp2/0, and Chinese hamster ovary (CHO)K1 cells were obtained from the American Type Culture Collection(ATCC, Manassas, VA). These cell lines were cultured at 37 °C in ahumidiﬁed atmosphere of 5% CO2 and 95% air in RPMI 1640 medium (Wako Pure Chemical Industries Ltd., Osaka, Japan), including
2 mM L-glutamine (Wako Pure Chemical Industries Ltd.) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life
Technologies Corp., Carlsbad, CA). One glioma patient (anaplastic
astrocytoma, WHO grade III), who underwent primary surgery at
Tohoku University Hospital and one glioma patient (glioblastoma,
WHO grade IV), who underwent primary surgery at Tsukuba University Hospital, were included in this study. Informed consent
was obtained from each patient or the patient’s guardian for
obtaining samples and for subsequent data analysis.
2.2. Hybridoma production
One mouse monoclonal antibody, RMab-22 against IDH2-wild
type and one rat monoclonal antibody against Maltose-binding
protein (MBP) were produced using traditional spleen method[13]. Brieﬂy, BALB/c mice or SD rats (CLEA Japan Inc., Tokyo, Japan)
were immunized by i.p. injections of 30 lg of synthetic peptide
CKPITIGKHAHGD (IDH2-R172K peptide), corresponding to aminoacids 166–177 of human IDH2-R172K plus N-terminus cysteine
conjugated with KLH or 100 lg of recombinant MBP protein,respectively, together with Imject Alum (Thermo Scientiﬁc Inc.,
Rockford, IL). One week later, secondary i.p. immunization of
30 lg of IDH2-R172K peptide or 100 lg of recombinant MBP protein was performed. After several additional immunization of
30 lg of IDH2-R172K peptide or 100 lg of recombinant MBP protein, a booster injection was given i.p. 2 days before spleen cells
were harvested. The spleen cells were fused with mouse myeloma
P3U1 cells using Sendai virus (Hemagglutinating Virus of Japan:

41

HVJ) envelope: GenomONE-CF (Ishihara Sangyo Kaisha Ltd., Osaka,
Japan) according to the manufacturer’s instructions [11,13,14]. The
hybridomas were grown in RPMI medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Life Technologies Corp.). The culture supernatants were screened using
ELISA for binding to the both IDH2-R172K and IDH2-wild type
(IDH2-WT) peptides, as described previously [13]. Two rat monoclonal antibodies (KMab-1 and MMab-1) against IDH2-mutations
and one mouse monoclonal antibody (KrMab-3) against IDH2WT were produced using rat or mouse medial iliac lymph node
methods [16]. Brieﬂy, WKY/Izm rats and BDF1 mice (Japan SLC
Inc., Shizuoka, Japan) were immunized by injecting 170 lg or
80 lg of synthetic peptides of CGGTKPITIGKHAHGDQYKA (IDH2R172K) and CGGTKPITIGMHAHGDQYKA (IDH2-R172M), conjugated with KLH together with Freund’s complete adjuvant (FCA;
Sigma–Aldrich, St. Louis, MO) into footpad or tailbase, respectively.
The lymphocytes were fused with mouse myeloma Sp2/0 cells
using polyethylene glycol (PEG) methods. The culture supernatants
were screened using ELISA for binding to the IDH2-mutations(IDH2-R172K and IDH2-R172M) and IDH2-WT peptides, conjugated with BSA.
2.3. Enzyme-Linked Immunosorbent Assay (ELISA)
Synthetic peptides were immobilized, respectively, on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientiﬁc Inc.,
Waltham, MA) at 1 lg/ml for 30 min, respectively. After blocking
with 1% BSA (Sigma–Aldrich) in PBS, the plates were incubatedwith culture supernatant, followed by 1:1000 diluted peroxidaseconjugated anti-mouse IgG (Dako, Glostrup, Denmark). The enzymatic reaction was conducted with a substrate solution containing
TMB (Thermo Fisher Scientiﬁc Inc.). After the reaction was stopped
with 2 M H2SO4, the optical density was measured at 450 nm witha Benchmark microplate reader (Bio-Rad Laboratories Inc., Philadelphia, PA). These reactions were performed with a volume of
50 ll at 37 °C.
2.4. Plasmid preparation
Human IDH2 cDNA (Accession No. NM_002168) encoding a fulllength open reading frame (ORF) was obtained by PCR using a
cDNA derived from the U373 glioblastoma cell line [17] as a template. The primer set for IDH2 was as follows: EcoRI-IDH2.F1, 50 ccGAATTCgggATGGCCGGCTACCTGCGGG-30
and
SalI-IDH2wterR1359, 50 -gccgtcgacCTACTGCCTGCCCAGGGCTCT-30 . The ampliﬁed cDNA was subcloned into a pcDNA3.1/V5-His-TOPO vector(Life Technologies Corp.). Substitution of the Arginine 172 (R172)
to appropriate amino acid codons (Lysine, Methionine, or Tryptophan) in IDH2 cDNAs was accomplished using the QuikChangeLightning site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). The full-length IDH2-WT, IDH2-R172K, IDH2-R172M,
and IDH2-R172W were subcloned into an expression vector,
pMAL-c2 (New England Biolabs Inc., Beverly, MA), via EcoRI and
SalI restriction sites. The full-length IDH2 and each mutated ORF
were ampliﬁed using the primer set: EcoRI-IDH2.F1 and IDH2woterR1356-XhoI,
50 taaCTCGAGcgCTGCCTGCCCAGGGCTCTG-30 .
These PCR products were digested with EcoRI and XhoI restriction
enzymes, and subcloned into pcDNA3 vector (Life Technologies
Corp.) together with the nucleotide sequence (ctcgagTGGCGTTGCCATGCCA GGTGCCGAAGATGATGTGGTGTAAtctaga), which encodes
12 amino acids, GVAMPGAEDDVV (PA tag).
2.5. Protein expression using bacteria cells and mammalian cells
Competent Escherichia coli TOP-10 cells (Life Technologies
Corp.) were transformed with the plasmid, pMAL-IDH2-WT,

42

M.K. Kaneko et al. / Biochemical and Biophysical Research Communications 432 (2013) 40–45

pMAL-IDH2-R172K, pMAL-IDH2-R172M, and pMAL-IDH2-R172W.
Then, they were cultured overnight at 37 °C in LB medium (LifeTechnologies Corp.) containing 100 lg/ml ampicillin (Sigma–Aldrich). Cell pellets were resuspended in phosphate buffered saline(PBS) with 1% Triton X-100 with 50 lg/ml aprotinin (Sigma–Aldrich). After sonication, the crude extracts were collected by centrifugation (9000g 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 Tris–HCl,
pH 7.4, 200 mM NaCl, 1 mM EDTA), and the fusion proteins were
eluted by column buffer with 10 mM maltose. CHO cells weretransfected with appropriate amounts of plasmids, pcDNA3/
IDH2-WT,pcDNA3/IDH2-R172K,
pcDNA3/IDH2-R172M,
or
pcDNA3/IDH2-R172W using Lipofectamine LTX (Life Technologies
Corp.) according to the manufacturer’s instructions. Stable transfectants were selected by cultivating the transfectants in medium
containing 1 mg/ml of G418 (Wako Pure Chemical Industries
Ltd.). The expression level of IDH2 was conﬁrmed using Westernblot analysis.
2.6. Western-blot analyses
Cultured cell pellets were lysed with PBS with 1% TritonX-100
for 30 min on ice. The lysate supernatants were centrifuged for
15 min at 15,000 rpm to remove cellular debris. Cell lysates containing 10 lg of total protein were prepared for Western-blot analysis by boiling in SDS sample buffer (Nakalai Tesque, Inc., Kyoto,
Japan). They were electrophoresed on 5–20% polyacrylamide gels
(Wako Pure Chemical Industries Ltd.). The separated proteins weretransferred to a PVDF membrane (Bio-Rad Laboratories Inc.). After
blocking with 4% skim milk in PBS for 15 min, the membrane wasincubated with KMab-1 (5 lg/ml), MMab-1 (1 lg/ml), RMab-22(5 lg/ml), KrMab-3 (5 lg/ml), TMab-2 (anti-MBP tag; 1 lg/ml),
or NZ-1 (anti-PA tag; 1 lg/ml) for 15 min; then with peroxidaseconjugated secondary antibodies (Dako, 1/1000 diluted) for
15 min, and developed with ECL-plus reagents (GE Healthcare,
Buckinghamshire, UK) using SAYAKA-IMAGER (DRC Co. Ltd., Tokyo,
Japan).
2.7. Immunocytochemical (ICC) analyses
Cultured cells were ﬁxed with 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4) for 20 min at room temperature, andpermeabilized with 0.1% Triton X-100 in PBS for 15 min. Cells werethen treated with 10% normal goat serum in PBS (NGS/PBS) to
block nonspeciﬁc binding sites, and were incubated with 5 lg/mlof MMab-1, RMab-22, KrMab-3, or anti-PA tag (NZ-1) containing
0.1% Triton X-100 overnight at 4 °C in a moist chamber. They wereincubated with goat anti-mouse or rat IgG-Alexa 488 (dilution
1:400; Molecular Probes Inc., Eugene, OR) in PBS containing 0.1%
Triton X-100 for 1 h at room temperature. Cells were also treatedwith TO-PRO-3 (Molecular Probes) to stain the cell nuclei. They
were examined using confocal laser-scanning microscopy (LSM5PASCAL; Carl Zeiss Inc., Jena, Germany). Then ﬂuorescent images
were processed using image-processing software (Adobe Photoshop; Adobe Systems Inc.).

Fig. 1. Western-blot analyses by anti-IDH2 mAbs against MBP-fusion proteins.
Puriﬁed proteins (0.05 lg/lane) of E. coli-expressing IDH2 wild type (WT; lane 1)
and IDH2 mutants (lane 2, IDH2-R172K; lane 3, IDH2-R172M; lane 4, IDH2-R172W)
were electrophoresed under reducing conditions using 5–20% gels, and wereWestern-blotted with KMab-1, MMab-1, RMab-22, KrMab-3, and anti-MBP tag(TMab-2).

2.8. Immunohistochemical (IHC) analysis
IDH2-R172M protein expression was determined immunohistochemically in parafﬁn-embedded tumor specimens. Brieﬂy, 4lm-thick histologic sections were deparafﬁnized in xylene andrehydrated. Then, they were autoclaved in citrate buffer (pH 6.0)
for 20 min. Sections were incubated with 10 lg/ml of MMab-1
for 1 h at room temperature. Biotin-conjugated secondary antirat IgG (Dako) was incubated for 30 min at room temperature followed by the peroxidase-conjugated biotin-streptavidin complex(Vectastain ABC Kit; Vector Laboratories Inc., Burlingame, CA) for
30 min at room temperature. Color was developed using 3, 3-diaminobenzidine tetrahydrochloride (DAB) for 10 min, and counterstained with hematoxylin.
3. Results and discussion
3.1. Production of mutated IDH2-speciﬁc antibodies
The IDH2 gene at 15q26.1 encodes isocitrate dehydrogenase 2(IDH2), which catalyzes the oxidative carboxylation of isocitrate
to a-ketoglutarate in mitochondria [18]. IDH2 mutations occur insome malignant gliomas [2], cartilaginous tumors [5,6], and inacute myeloid leukemias [8]. To date, almost all IDH2 mutations
have been identiﬁed at the critical isocitrate binding site R172 co-Table 1
Reactivities of anti-IDH2 monoclonal antibodies against antigens.
AntigenClone

Speices

Subclass

ELISA

WB (E. coli)

WB (CHO)ICC

IDH2-R172K
IDH2-R172M
IDH2-wild type
IDH2-wild type

KMab-1MMab-1RMab-22
KrMab-3

Rat
Rat
Mouse
Mouse

IgG2b, kappa
IgG2b, kappa
IgG2b, kappa
IgG2b, kappa

+++
+++
+++
+++

+++
+++
+++
+++

+
+++
++
++


+++
++
++

+++: high reactivity; ++: medium reactivity; +: low reactivity; : negative.

M.K. Kaneko et al. / Biochemical and Biophysical Research Communications 432 (2013) 40–45

don and R140 codon. The vast majority of changes of IDH2 are heterozygous in the same way of IDH1 mutation [1–4]. These discoveries mark unique ﬁndings in the ﬁeld of cancer genetics.
We have established three anti-mutated IDH1 mAbs: IMab-1[14] or HMab-1 [11] against IDH1-R132H and SMab-1 [13] against
IDH1-R132S. Hartmann et al. reported that IDH1 mutations include
R132H (664/716: 92.7%), R132C (29/716: 4.2%), R132S (11/716:
1.5%), R132G (10/716: 1.4%), and R132L (2/716: 0.2%); therefore,
combination of HMab-1 and SMab-1 cover 94.2% of IDH1 mutations. However, speciﬁc antibodies against IDH2 mutations havenot been reported. Hartmann et al. also reported that IDH2 mutations include R172K (20/31: 64.5%), R172M (6/31: 19.3%), andR172W (5/31: 16.2%) in gliomas [19]. For this study, we immunized mice or rats with synthetic peptides of several IDH2 mutants
including R172K and R172M. After cell fusion, the wells of hybridomas, which produced IDH2-R172K-reactive/IDH2-wild type(WT)-nonreactive or IDH2-R172M-reactive/IDH2-WT-nonreactive
were screened in ELISA. After limiting dilution, clone KMab-1 (rat
IgG2b subclass) against IDH2-R172K and clone MMab-1 (rat IgG2b
subclass) against IDH2-R172M were established (Table 1). Twoanti-IDH2-WT mAbs, clone RMab-22 (mouse IgG2b) and clone
KrMab-3 (mouse IgG2b), were also established from R172K-immunized mice (Table 1). KMab-1 and MMab-1 were shown to be each
IDH2 mutation-speciﬁc mAbs, not reacting with IDH2-WT, using
ELISA against both synthetic peptides and recombinant proteins(data not shown). In contrast, RMab-22 and KrMab-3 were shown
to be reactive with both IDH2-R172K and IDH2-WT using ELISA
against both synthetic peptides and recombinant proteins (datanot shown).

43

3.2. Speciﬁcity of anti-mutated IDH2 mAbs against IDH2 mutants
To determine the speciﬁcity of KMab-1, MMab-1, RMab-22, andKrMab-3 mAbs, the reactivity against IDH2-WT and IDH2 mutants(MBP-IDH2-R172K, MBP-IDH2-R172M, MBP-IDH2-R172W) were
investigated using Western-blot analyses. Fig. 1 shows that
KMab-1 recognized only IDH2-R172K protein, not the other proteins (IDH2-WT, IDH2-R172M, IDH2-R172W), indicating that
KMab-1 is a speciﬁc antibody against IDH2-R172K protein. In contrast, MMab-1 reacted with only IDH2-R172M protein, not theother proteins (IDH2-WT, IDH2-R172K, IDH2-R172W), indicatingthat MMab-1 is a speciﬁc antibody against IDH2-R172M protein.
RMab-22 recognized IDH2-WT, IDH2-R172K, and IDH2-R172M
proteins, not IDH2-R172W protein. In contrast, KrMab-3 recognized all IDH2 mutants, although the reactivity against IDH2R172W is comparatively weak. Both RMab-22 and KrMab-3 were
produced from IDH2-R172K-immunized mice; therefore, bothmAbs might be able to react strongly with IDH2-R172K comparedwith IDH2-R172M or IDH2-R172W. These results indicate that
KMab-1 and MMab-1 are useful in detecting IDH2-R172K or
IDH2-R172M, respectively.
We next performed Western-blot analyses against mutated
IDH2-expressing CHO cells. As shown in Fig. 2A, KMab-1 recognized only IDH2-R172K protein expressed in CHO cells, not theother proteins (IDH2-WT, IDH2-R172M, IDH2-R172W). MMab-1
recognized only IDH2-R172M protein expressed in CHO cells, notthe other proteins (IDH2-WT, IDH2-R172K, IDH2-R172W). These
results indicate that KMab-1 and MMab-1 are useful in detecting
IDH2-R172K or IDH2-R172M expressed in mammalian cells,

Fig. 2. The speciﬁcity of anti-mutated IDH2 mAbs. (A) Western-blot analyses by anti-IDH2 mAbs against mutated IDH2-expressing CHO cells. Total cell lysate from CHO cells
expressing IDH2 wild type (WT; lane 1) and IDH2 mutants (lane 2, IDH2-R172K; lane 3, IDH2-R172M; lane 4, IDH2-R172W) were electrophoresed under reducing conditions
using 5–20% gels, and were Western-blotted with KMab-1, MMab-1, and anti-PA tag (NZ-1). (B) Immunocytochemical analysis by anti-mutated IDH2 mAbs against mutated
IDH2-transfected CHO cells. CHO cells were transfected by mutated IDH2. Each cell was stained by MMab-1 or anti-PA tag (NZ-1) followed by anti-rat IgG-Alexa 488 antibody(green). Cells were also treated with TO-PRO-3 to stain the cell nuclei (blue). (C and D) Immunohistochemical analysis by MMab-1 against one glioma tissue. Glioma tissue
having IDH2-R172M was stained with MMab-1. Magniﬁcation: 400. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web
version of this article.)44

M.K. Kaneko et al. / Biochemical and Biophysical Research Communications 432 (2013) 40–45

did not react with IDH2-R172K in ICC, dissociation constant(KD) was determined using ELISA method [20]. As a result, KD of
KMab-1 was determined to be 1.5  107 M against recombinant
protein of IDH2-R172K, whereas that of KMab-1 was
2.8  109 M against synthetic peptide IDH2-R172K, indicatingthat the binding-afﬁnity of KMab-1 is extremely low against
IDH2-R172K protein. Afﬁnity maturation of KMab-1 should benecessary to increase the binding afﬁnity of KMab-1 against
IDH2-R172K protein. RMab-22 and KrMab-3 reacted with IDH2WT-expressing CHO cells, suggesting that both RMab-22 andKrMab-3 are also useful in ICC (Table 1). Next, we investigated
the speciﬁcity of MMab-1 against mutated IDH2-expressing CHO
cells. As depicted in Fig. 2B, MMab-1 reacted with only CHO/
IDH2-R172M, not with CHO/IDH2-WT and CHO/IDH2-R172W.
Immunostaining of MMab-1 for CHO/IDH2-R172M demonstrated
cytosolic patterns. These results indicate that MMab-1 is useful indetecting IDH2-R172M, suggesting that MMab-1 could be also useful in immunohistochemical analyses for detection of IDH2-R172M.
In the same way with Western-blot analyses, both RMab-22 andKrMab-3 reacted with IDH2-WT and IDH2-R172K (Fig. 3B).
3.4. Immunohistochemical (IHC) analysis by MMab-1 against IDH2R172M-bearing gliomas

Fig. 3. The speciﬁcity of anti-IDH2-wild type (WT) mAbs. (A) Western-blot analyses
by anti-IDH2 mAbs against mutated IDH2-expressing CHO cells. Total cell lysate
from CHO cells expressing IDH2-WT (lane 1) and IDH2 mutants (lane 2, IDH2R172K; lane 3, IDH2-R172M; lane 4, IDH2-R172W) were electrophoresed under
reducing conditions using 5–20% gels, and were Western-blotted with RMab-22,
KrMab-3, and anti-PA tag (NZ-1). (B) Immunocytochemical analysis by anti-IDH2
mAbs against mutated IDH2-transfected CHO cells. CHO cells were transfected by
IDH2-WT and IDH2-R172K. Each cell was stained by RMab-22 and KrMab-3,
followed by anti-mouse IgG-Alexa 488 antibody (green). Cells were also treatedwith TO-PRO-3 to stain the cell nuclei (blue). (For interpretation of the references tocolor in this ﬁgure legend, the reader is referred to the web version of this article.)

We next performed immunohistochemistry of MMab-1 against
IDH2-R172M-positive gliomas. Typical results are shown in Fig. 2C
and D. MMab-1 stained almost all tumor cells of IDH2-R172M-positive gliomas (Fig. 2C), although no staining was observed in
IDH2-WT gliomas (data not shown). Further, MMab-1 did not stain
endothelial cells in gliomas (Fig. 2D). These results indicate that
MMab-1 is useful in immunohistochemical analyses for detectionof IDH2-R172M mutation. In near future, combination of MMab1 with previously established anti-mutated IDH1 mAbs (IMab-1,
HMab-1, and SMab-1) should increase the diagnostic rate using
immunohistochemistry against glioma patients.
In conclusion, the established anti-mutated IDH2-speciﬁc mAbs
are expected to be extremely useful for diagnosis and biologicalevaluation of mutation-bearing gliomas in combination with
anti-mutated IDH1-speciﬁc monoclonal antibodies. Additional
IDH1/2 mutation-speciﬁc antibodies such as anti-IDH1-R132G,
anti-IDH1-R132L, or anti-IDH1-R132C must be developed for pathological diagnosis of gliomas, cartilaginous tumors, and AML.
Acknowledgments

respectively. As shown in Fig. 3A, both RMab-22 and KrMab-3
strongly recognized only IDH2-WT and IDH2-R172K. Although
KrMab-3 detects both endogenous hamster IDH2 and exogenous
human IDH2 in cell lysate from CHO/IDH2-R172M and CHO/
IDH2-R172W, RMab-22 slightly reacts with only endogenous hamster IDH2 in cell lysate from CHO/IDH2-R172M and CHO/IDH2R172W. Because an anti-PA tag antibody (NZ-1) equally reacts
with all mutated IDH2 proteins from each cell lysate, RMab-22
and KrMab-3 are able to recognize IDH2-R172K more strongly than
IDH2-R172M and IDH2-R172W.
3.3. Immunocytochemical (ICC) analyses by anti-mutated IDH2 mAbs
against mutated IDH2-expressing cell lines
Newly established anti-IDH2 mAbs, KMab-1 and MMab-1 wereconﬁrmed to be anti-mutated IDH2 speciﬁc antibodies in Westernblot analyses; therefore, we performed immunocytochemical (ICC)
analyses using IDH2-R172K- or IDH2-R172M-transfected CHO
cells. Results show that MMab-1 reacted with IDH2-R172M-transfected cells; however, the reaction of KMab-1 against IDH2-R172K
was not observed (Table 1). To investigate the reason why KMab-1

We thank Sachiko Suzuki and Junko Aita for their excellent
technical assistance. This work was supported in part by Platformfor Drug Discovery, Informatics, and Structural Life Science (M.K.K.,
Y.K.), by Regional Innovation Strategy Support Program (Y.K.), andby KAKENHI (23790185, 23701043): a Grant-in-Aid for Young Scientists (B) (M.K.K., Y.K.) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan, by Intelligent Cosmos
Academic Foundation (Y.K.), and by Ofﬁce for Gender Equality of
Yamagata University (M.K.K.).
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