Biochemical and Biophysical Research Communications 466 (2015) 733e739

Contents lists available at ScienceDirect

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

A high-sensitive HMab-2 speciﬁcally detects IDH1-R132H, the mostcommon IDH mutation in gliomas
Yuki Fujii a, 1, Satoshi Ogasawara a, 1, Hiroharu Oki a, 1, Xing Liu a, Mika K. Kaneko a,
Shingo Takano b, Yukinari Kato a, *ab

Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
Department of Neurosurgery, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 3 September 2015
Accepted 12 September 2015
Available online 14 September 2015

Isocitrate dehydrogenase 1 (IDH1) mutations have been detected in gliomas and other tumors. Although
IDH1 catalyzes the oxidative carboxylation of isocitrate to a-ketoglutarate (a-KG) in cytosol, mutated
IDH1 proteins possess the ability to change a-KG into the oncometabolite D-2-hydroxyglutarate (D-2HG).
Several monoclonal antibodies (mAbs) speciﬁc for IDH1 mutations have been established, such as H09,
IMab-1, and HMab-1 against IDH1-R132H, which is the most frequent IDH1 mutation in gliomas. In thisstudy, we established a novel high-sensitive mAb HMab-2, which reacts with IDH1-R132H but not withwild type IDH1 in ELISA. HMab-2 reacted only with IDH1-R132H, not with wild type IDH1/2 and other
IDH1/2 mutants in Western-blot analysis. Furthermore, HMab-2 recognized IDH1-R132H more sensitively compared with our previously established HMab-1. HMab-2 detected endogenous IDH1-R132H
protein expressed in glioblastoma in immunohistochemical analysis. HMab-2 is expected to be useful forthe diagnosis of IDH1-R132H-bearing tumors.
© 2015 Elsevier Inc. All rights reserved.

Keywords:
IDH1
IDH1 mutation
R132H
Monoclonal antibody
HMab-2

1. Introduction
Isocitrate dehydrogenase (IDH) 1 and IDH2 catalyze the oxidative carboxylation of isocitrate to a-ketoglutarate (a-KG) in thecytosol and mitochondria, respectively [1]. IDH1/2 mutations havebeen found to change a-KG into the oncometabolite D-2hydroxyglutarate (D-2HG). IDH1/2 mutations occur in some gliomas [2e7], acute myeloid leukemias [8], intrahepatic cholangiocarcinomas [9], and cartilaginous tumors [10]. In
astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary glioblastomas, IDH1/2 mutations have been identiﬁed as
early and frequent genetic alterations (50e90%) [11]. In contrast,
primary glioblastomas rarely contain IDH1/2 mutations (3e16%).
The vast majority of IDH1/2 mutations are heterozygous, and IDH1
mutations and IDH2 mutations are mutually exclusive [12]. IDH1
mutations are remarkably speciﬁc to a single codon in theconserved and functionally important arginine 132 residue (R132).

Abbreviations: IDH, isocitrate dehydrogenase; mAb, monoclonal antibody.
* Corresponding author.
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.
http://dx.doi.org/10.1016/j.bbrc.2015.09.070
0006-291X/© 2015 Elsevier Inc. All rights reserved.

IDH2 mutations are speciﬁc to a single codon in arginine 172 residue (R172) in gliomas; however, in AML, IDH2 mutations in arginine 140 residue (R140) have been discovered and were foundmore frequently than R172 [13e15]. In gliomas, previously reported
IDH1/2 mutations include IDH1-R132H (89.7%), IDH1-R132C (2.8%),
IDH2-R172K (2.8%), IDH1-R132S (1.4%), IDH1-R132G (1.1%), IDH2R172M (0.8%), IDH1-R132L (0.6%), IDH2-R172W (0.6%), and IDH2R172S (0.2%) [12]. In the present study, we report a novel highsensitive mAb HMab-2, which reacts with IDH1-R132H but notwith wild type IDHs and other mutated IDHs.
2. Materials and methods
2.1. Cell lines and specimens
P3X63Ag8U.1 (P3U1) mouse myeloma cell line and LN229
glioblastoma cell line were purchased 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. P3U1 wascultured in Roswell Park Memorial Institute (RPMI) 1640 medium(Nacalai Tesque Inc., Kyoto, Japan), and LN229 was cultured in
Dulbecco's modiﬁed Eagle medium (DMEM; Nacalai Tesque Inc.).
Both media include 2 mM L-glutamine (Nacalai Tesque Inc.), 100

734

Y. Fujii et al. / Biochemical and Biophysical Research Communications 466 (2015) 733e739

units/ml of penicillin, 100 mg/ml of streptomycin, and 25 mg/ml ofamphotericin B (Nacalai Tesque, Inc.) and 10% heat-inactivated fetal
bovine serum (FBS; Thermo Fisher Scientiﬁc Inc., Waltham, MA).
Two patients who underwent primary surgery at Tsukuba University Hospital are included in this study. Informed consent was obtained from each patient or the patient's caretaker for obtaining
samples and subsequent data analysis.
2.2. Hybridoma production
BALB/c mice were immunized by intraperitoneal (i.p.) injectionsof 100 mg of synthetic peptides of CKPIIIGHHAYGD (IDH1-R132H),
conjugated with keyhole limpet hemocyanin (KLH) together with
Imject Alum (Thermo Fisher Scientiﬁc Inc.). One week later, secondary i.p. immunization of 100 mg of synthetic peptides was performed. After several additional immunizations of 100 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 RPMI medium with hypoxanthine,aminopterin, and thymidine (HAT) selection medium supplement(Thermo Fisher Scientiﬁc Inc.). The culture supernatants werescreened using enzyme-linked immunosorbent assay (ELISA).
2.3. Enzyme-linked immunosorbent assay (ELISA)
Recombinant proteins were immobilized on Nunc Maxisorp 96-

well immunoplates (Thermo Fisher Scientiﬁc Inc.) at 1 mg/ml with avolume of 50 ml at 37  C for 30 min. After blocking with 1% BSA in
phosphate buffered saline (PBS) with a volume of 200 ml at 4  C overnight, the plates were incubated with culture supernatant with avolume of 50 ml at 37  C for 30 min with subsequent 1:3000 diluted
peroxidase-conjugated anti-mouse IgG (Dako, Glostrup, Denmark) in
0.1% BSA/PBS with a volume of 50 ml at 37  C for 30 min. The enzymatic reaction was conducted with 1-Step Ultra TMB-ELISA (Thermo
Fisher Scientiﬁc Inc.) with a volume of 50 ml at room temperature for10 min. The optical density was measured at 655 nm using an iMark
microplate reader (Bio-Rad Laboratories Inc., Philadelphia, PA).
2.4. Protein expression using bacteria cells
Competent Escherichia coli TOP-10 cells (Thermo Fisher Scientiﬁc Inc.) were transformed with appropriate amounts of plasmids,
pMAL-IDH1-WT or pMAL-IDH1-R132H [16]. Then, they were
cultured overnight at 37  C in LB medium (Thermo Fisher ScientiﬁcInc.) containing 100 mg/ml ampicillin (SigmaeAldrich Corp.). Cell
pellets were resuspended in PBS with 1% Triton X-100 with 50 mg/
ml aprotinin (SigmaeAldrich Corp.). After sonication using Branson
Advanced Soniﬁer (Branson Ultrasonics Corp., Danbury, CT), thecrude extracts were collected by centrifugation (9000  g, 30 min,4  C). The supernatants were loaded onto amylose resin (New England Biolabs Inc., Ipswich, MA). The loaded resins were washedextensively with column buffer (20 mM Tris-HCl, pH 7.4, 200 mM
NaCl, 1 mM EDTA). The fusion proteins were eluted by column

Fig. 1. (A) Western-blot analyses by anti-IDH1/2 mAbs against IDH1-wild type or IDH1-R132H-expressing LN229 cells. Cell lysates were prepared for Western blot analyses byboiling in SDS sample buffer. They were electrophoresed on 5e20% polyacrylamide gels. The separated proteins were transferred to a PVDF membrane. After blocking, themembrane was incubated with HMab-2 (1 mg/ml), HMab-1 (5 mg/ml), OGHis (anti-His tag; 1 mg/ml), RMab-3 (1 mg/ml), and AC-15 (anti-b-actin; 1 mg/ml). Then the membrane wasincubated with peroxidase-conjugated anti-mouse antibody, and developed with ImmunoStar LD Chemiluminescence Reagent using Sayaca-Imager. Red arrow, exogenous IDH1;
black arrow, endogenous IDH1. (B) Western-blot analyses by anti-IDH1/2 mAbs against mutated IDH1/2-expressing LN229 cells. The membrane was incubated with HMab-2 (1 mg/ml), OGHis (anti-His tag; 1 mg/ml), RcMab-1 (1 mg/ml), NZ-1 (anti-PA tag; 1 mg/ml), 5F11 (1 mg/ml), and AC-15 (anti-b-actin; 1 mg/ml). Red arrow, exogenous IDH1; black arrow,endogenous IDH1; blue arrow, exogenous IDH2; green arrow, endogenous IDH2. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web
version of this article.)Y. Fujii et al. / Biochemical and Biophysical Research Communications 466 (2015) 733e739

buffer with 10 mM maltose.
2.5. Protein expression using mammalian cells
LN229 cells were transfected with appropriate amounts ofplasmids, pcDNA3.1V5tag-Histag (VH)/IDH1-WT, pcDNA3.1VH/
IDH1-R132H, pcDNA3.1VH/IDH1-R132C, pcDNA3.1VH/IDH1-R132S,
pcDNA3.1VH/IDH1-R132G, pcDNA3.1VH/IDH1-R132L, pcDNA3PAtag (PA)C-terminal(c)high-binding(H)/IDH2-WT, pcDNA3-PAcH/
IDH2-R172K, pcDNA3-PAcH/IDH2-R172M, pcDNA3-PAcH/IDH2R172W, pcDNA3-PAcH/IDH2-R172S, pcDNA3-PAcH/IDH2-R172G

735

using Lipofectamine LTX (Thermo Fisher Scientiﬁc Inc.) according
to the manufacturer's instructions [17]. The expression level of
IDH1/2 was conﬁrmed using Western blot analyses.
2.6. 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 by boiling in SDS sample buffer(Nacalai Tesque, Inc.). They were electrophoresed on 5e20%

Fig. 2. Immunohistochemical analysis by HMab-2. Sections of IDH1-wild type glioblastoma (AeD) and IDH1-R132H-posssesing glioblastoma (EeH) were incubated with 2 mg/ml of
HMab-2 (B, D, F, H), followed by Envision þ kit, and color was developed using DAB and counterstained with hematoxylin. Serial sections were also stained by hematoxlylin-eosin(A, C, E, G). Scale bar: 100 mm. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

736

Y. Fujii et al. / Biochemical and Biophysical Research Communications 466 (2015) 733e739

polyacrylamide gels (Wako Pure Chemical Industries Ltd., Osaka,
Japan). 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 HMab-2, HMab-1 [18], RMab-3 [18], RcMab-1[19], 5F11 (anti-IDH2; SigmaeAldrich Corp.), OGHis (anti-His tag;
MBL Co. Ltd., Nagoya, Japan), NZ-1 (anti-PA tag) [20,21], and AC-15(anti-b-actin; SigmaeAldrich Corp.) for 60 min. Then the membrane 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.) using Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
2.7. Immunohistochemical analysis
IDH1-R132H protein expression was determined immunohistochemically in parafﬁn-embedded tumor specimens, as described
previously [22,23]. Brieﬂy, 5-mm-thick histologic sections weredeparafﬁnized in xylene and rehydrated. Then they were autoclavedin citrate buffer (pH 6.0; Dako) for 20 min. Sections were incubatedwith 2 mg/ml of HMab-2, 5 mg/ml of RMab-3, 5 mg/ml of RcMab-1,
2 mg/ml of isotype control (clone PMab-2; mouse IgG1, kappa), or
PBS overnight at 4  C. Blocking peptides were used at a concentration of 20 mg/ml. The Envision þ kit (Dako) or biotinylated anti-rat
IgG (Dako) þ Vectastain Elite ABC kit (Vector laboratories Inc.,
Burlingame, CA) was used for secondary mAbs and ampliﬁcation.
Color was developed using 3, 3-diaminobenzidine tetrahydrochloride (DAB) for 10 min, and counterstained with hematoxylin.
3. Results
3.1. Production of a mutated IDH1-R132H-speciﬁc mAb
We immunized mice with synthetic peptides of IDH1-R132H,and screened IDH1-R132H-reactive/IDH1-wild type (WT)-nonreactive mAbs using ELISA. After limiting dilution, a clone HMab-2(mouse IgG1, kappa) against IDH1-R132H was established. HMab-2
also reacted with MBP-fused IDH1-R132H protein, not with MBPfused IDH1-WT protein using Western-blot analysis (data notshown). We next investigated the reactivity of HMab-2 against
IDH1-R132H, which is expressed in mammalian cells in Westernblot analysis. HMab-2 detected IDH1-R132H, which is transfectedin LN229 cells, whereas it did not react with IDH1-WT, which istransfected in LN229 cells or endogenous IDH1 (Fig. 1A). Previous
established HMab-1, which is very useful in immunohistochemistry[18,24], recognized IDH1-R132H at a concentration of 5 mg/ml. In
contrast, HMab-2 detected IDH1-R132H at a concentration of 1 mg/
ml. Furthermore, the signal intensity of HMab-2 at a concentrationof 1 mg/ml was stronger than that of HMab-1 at a concentration of
5 mg/ml. RcMab-1 (anti-IDH) detected both exogenous andendogenous IDH1 proteins. OGHis (anti-His tag) reacted withexogenous IDH1 proteins.
3.2. Speciﬁcity of HMab-2 against IDH proteins in Western blot
analyses
To determine the speciﬁcity of HMab-2, we performed Westernblot analyses against IDH1/2 mutations, which are expressed in
LN229 glioblastoma cells. IDH1/2-WTs and IDH1/2 mutants (IDH1R132H, IDH1-R132C, IDH1-R132S, IDH1-R132G, IDH1-R132L,
IDH2-R172K, IDH2-R172M, IDH2-R172W, IDH2-R172S, IDH2-R172G)
were transfected in LN229, and Western-blotted with anti-IDH
mAbs. As shown in Fig. 1B, RcMab-1 detected both exogenous andendogenous IDH1 proteins. 5F11 reacted with both exogenous andendogenous IDH2 proteins. Anti-tag mAbs, OGHis (anti-His tag) andNZ-1 (anti-PA tag) reacted with exogenous IDH1 and IDH2 proteins,respectively. Interestingly, HMab-2 speciﬁcally detected IDH1R132H, but did not react with IDH1/2-WTs and other mutations.

Fig. 3. HMab-2 reaction was blocked by immunized peptide in immunohistochemical analysis. The section of IDH1-R132H-posssesing glioblastoma was stained by hematoxlylineosin (A). Serial sections were incubated with 2 mg/ml of isotype control (B), 2 mg/ml of HMab-2 (C), or 2 mg/ml of HMab-2 þ 20 mg/ml of immunized peptide (D) followed byEnvision þ kit, and color was developed using DAB and counterstained with hematoxylin. Scale bar: 100 mm. (For interpretation of the references to colour in this ﬁgure legend, thereader is referred to the web version of this article.)Y. Fujii et al. / Biochemical and Biophysical Research Communications 466 (2015) 733e739

3.3. HMab-2 reacts with endogenous IDH1-R132H expressed inglioblastoma tissues
We selected two glioblastoma tissues: one possesses only
IDH1-WT (Fig. 2AeD); the other possesses IDH1-R132H, which ispreviously investigated using DNA direct sequencing (Fig. 2EeH)[25]. As shown in Fig. 2F and H, HMab-2 clearly stained IDH1R132H-possessing glioma cells, whereas it did not react with
vascular endothelial cells. In contrast, HMab-2 did not stain glioma

737

cells of IDH-WT tissue (Fig. 2 B and D). HMab-2 reaction wascompletely inhibited by the immunized peptide, demonstratingthat HMab-2 reaction is speciﬁc against IDH1-R132H protein ofglioma cells (Fig. 3). These data indicate that HMab-2 is speciﬁc
against IDH1-R132H of gliomas in immunohistochemical analyses.
Anti-IDH1 mAbs such as RMab-3 and RcMab-1 strongly reactedwith glioma cells of both IDH-WT tissue and IDH1-R132H tissue,
indicating that IDH1 expression was not suppressed in IDH-WT
tissue (Fig. 4).

Fig. 4. Anti-IDH1 mAbs strongly reacted with glioma cells of both IDH-WT tissue and IDH1-R132H tissue. The section of IDH1-wild type glioblastoma (AeD) and IDH1-R132Hposssesing glioblastoma (EeH) were incubated with 2 mg/ml of HMab-2 (A, E), 5 mg/ml of RMab-3 (B, F), 5 mg/ml of RcMab-1 (C, G), or PBS (D, H) followed by Envision þ kit (A, B, D,
E, F, H) or biotinylated anti-Rat IgG and Vectastain Elite ABC kit (C, G), and color was developed using DAB and counterstained with hematoxylin. Scale bar: 100 mm. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

738

Y. Fujii et al. / Biochemical and Biophysical Research Communications 466 (2015) 733e739

4. Discussion
There are several methods for testing IDH mutation status,
which targets DNA sequence, protein, and D-2-hydroxyglutarate(D-2HG) [26]. DNA direct sequencing (Sanger method) andimmunohistochemistry are two conventional methods, which areapplied for daily diagnostic practice. Recently, several special
methods such as melting curve analysis [27], pyrosequencing [28],and magnetic resonance spectroscopy (MRS) [29] were developed;
however, special equipment are required for those methods [26].
Although Sanger method detects all types of mutations, at least 20%of the mutant allele is required for detection of IDH mutations [28].
In contrast, immunohistochemistry detects only one mutationbearing tumor cell; however, many types of monoclonal antibodies (mAbs) against IDH mutations should be developed. We
have established two multi-speciﬁc IDH mAbs [24]: MsMab-1 [16]and MsMab-2 [19]. We also established several mono-speciﬁc antimutated IDH mAbs [24]: IDH1-R132H-speciﬁc mAbs (IMab-1 [30]and HMab-1 [18]), IDH1-R132S-speciﬁc mAb SMab-1 [31], IDH2R172K-speciﬁc mAb KMab-1 [32], IDH2-R172M-speciﬁc mAb
MMab-1 [32], and IDH2-R172W-speciﬁc mAb WMab-1 [33]. In
contrast, clone H09 against IDH1-R132H, which was established byanother group, has been used in many studies [34]. However, H09 iscross-reactive with IDH-R132L, and IMab-1 and HMab-1 are lowsensitive [24].
In the present study, we established a novel high-sensitive andhigh-speciﬁc mAb HMab-2, which reacts with IDH1-R132H but notwith IDH1-WT and other IDH mutants. Compared with our previous trials of producing anti-IDH1-R132H mAbs, we used threedifferent methods for the ﬁrst screening: i) the short peptide of 12
amino acids (þN-terminal cysteine) was used for immunization; ii)
the recombinant proteins of IDH1, which were fused with maltosebinding protein (MBP), were used for the ﬁrst ELISA screening
because recombinant protein-binding mAbs in ELISA have beenreported to be more advantageous in immunohistochemistry [24];iii) our previous studies used Chinese hamster ovary (CHO)-transfectants for the second screening, whereas LN229 glioblastomatransfectants were used in this study in anticipation of the direct
application of immunohistochemistry against gliomas. Previous
established HMab-1, which is very useful in immunohistochemistry[18,24], is unable to detect IDH1-R132H at a concentration of 1 mg/
ml in Western-blot, although HMab-2 can detect it at a concentration of less than 1 mg/ml. Furthermore, the signal intensity ofHMab-2 at a concentration of 1 mg/ml is stronger than that ofHMab-1 at a concentration of 5 mg/ml in immunohistochemistry,
demonstrating that HMab-2 is high-sensitive against IDH1-R132H.
Taken together, HMab-2 is expected to be useful for the diagnosis of
IDH1-R132H-bearing tumors, especially gliomas. HMab-2 is not
only high-sensitive compared with HMab-1 mAb but also highspeciﬁc compared with H09 mAb. The combination of HMab-2
with previously established anti-mutated IDH1/2 mAbs mightlead to high-sensitive detection of IDH1/2 mutation in clinicaldiagnosis.
Acknowledgments
We thank Takuro Nakamura, Noriko Saidoh, and Kanae Yoshida
for their excellent technical assistance. This work was supported inpart by the Practical Research for Innovative Cancer Control fromJapan Agency for Medical Research and Development, AMED (Y.K.);
by JSPS KAKENHI Grant Number 25462242 (Y.K.), by the Platformfor Drug Discovery, Informatics, and Structural Life Science (PDIS)
from AMED (Y.K.); by the Basic Science and Platform Technology
Program for Innovative Biological Medicine from AMED (Y.K.); andby the Regional Innovation Strategy Support Program from the

Ministry of Education, Culture, Sports, Science and Technology(MEXT) of Japan (Y.K.).
Transparency document
Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.bbrc.2015.09.070.
Author disclosure statement
The authors have no ﬁnancial interest to disclose.
Conﬂict of interest
None.
References[1] L. Dang, D.W. White, S. Gross, B.D. Bennett, M.A. Bittinger, E.M. Driggers,
V.R. Fantin, H.G. Jang, S. Jin, M.C. Keenan, K.M. Marks, R.M. Prins, P.S. Ward,
K.E. Yen, L.M. Liau, J.D. Rabinowitz, L.C. Cantley, C.B. Thompson, M.G. Vander
Heiden, S.M. Su, Cancer-associated IDH1 mutations produce 2hydroxyglutarate, Nature 462 (2009) 739e744.
[2] D.W. Parsons, S. Jones, X. Zhang, J.C. Lin, R.J. Leary, P. Angenendt, P. Mankoo,
H. Carter, I.M. Siu, G.L. Gallia, A. Olivi, R. McLendon, B.A. Rasheed, S. Keir,T. Nikolskaya, Y. Nikolsky, D.A. Busam, H. Tekleab, L.A. Diaz Jr., J. Hartigan,
D.R. Smith, R.L. Strausberg, S.K. Marie, S.M. Shinjo, H. Yan, G.J. Riggins,
D.D. Bigner, R. Karchin, N. Papadopoulos, G. Parmigiani, B. Vogelstein,
V.E. Velculescu, K.W. Kinzler, An integrated genomic analysis of human glioblastoma multiforme, Science 321 (2008) 1807e1812.
[3] H. Yan, D.W. Parsons, G. Jin, R. McLendon, B.A. Rasheed, W. Yuan, I. Kos,
I. Batinic-Haberle, S. Jones, G.J. Riggins, H. Friedman, A. Friedman, D. Reardon,
J. Herndon, K.W. Kinzler, V.E. Velculescu, B. Vogelstein, D.D. Bigner, IDH1 andIDH2 mutations in gliomas, N. Engl. J. Med. 360 (2009) 765e773.
[4] K. Ichimura, D.M. Pearson, S. Kocialkowski, L.M. Backlund, R. Chan, D.T. Jones,
V.P. Collins, IDH1 mutations are present in the majority of common adultgliomas but rare in primary glioblastomas, Neuro-oncology 11 (2009)341e347.
[5] C. Hartmann, J. Meyer, J. Balss, D. Capper, W. Mueller, A. Christians, J. Felsberg,
M. Wolter, C. Mawrin, W. Wick, M. Weller, C. Herold-Mende, A. Unterberg,
J.W. Jeuken, P. Wesseling, G. Reifenberger, A. von Deimling, Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas, Acta
Neuropathol. 118 (2009) 469e474.
[6] Y. Sonoda, T. Kumabe, T. Nakamura, R. Saito, M. Kanamori, Y. Yamashita,
H. Suzuki, T. Tominaga, Analysis of IDH1 and IDH2 mutations in Japaneseglioma patients, Cancer Sci. 100 (2009) 1996e1998.
[7] H. Arita, Y. Narita, S. Fukushima, K. Tateishi, Y. Matsushita, A. Yoshida,
Y. Miyakita, M. Ohno, V.P. Collins, N. Kawahara, S. Shibui, K. Ichimura, Upregulating mutations in the TERT promoter commonly occur in adult malignant
gliomas and are strongly associated with total 1p19q loss, Acta Neuropathol.
126 (2013) 267e276.
[8] A. Green, P. Beer, Somatic mutations of IDH1 and IDH2 in the leukemictransformation of myeloproliferative neoplasms, N. Engl. J. Med. 362 (2010)369e370.
[9] D.R. Borger, K.K. Tanabe, K.C. Fan, H.U. Lopez, V.R. Fantin, K.S. Straley,
D.P. Schenkein, A.F. Hezel, M. Ancukiewicz, H.M. Liebman, E.L. Kwak,
J.W. Clark, D.P. Ryan, V. Deshpande, D. Dias-Santagata, L.W. Ellisen, A.X. Zhu,
A.J. Iafrate, Frequent mutation of isocitrate dehydrogenase (IDH)1 and IDH2 incholangiocarcinoma identiﬁed through broad-based tumor genotyping,Oncologist 17 (2012) 72e79.
[10] M.F. Amary, S. Damato, D. Halai, M. Eskandarpour, F. Berisha, F. Bonar,
S. McCarthy, V.R. Fantin, K.S. Straley, S. Lobo, W. Aston, C.L. Green, R.E. Gale,R. Tirabosco, A. Futreal, P. Campbell, N. Presneau, A.M. Flanagan, Ollier disease
and Maffucci syndrome are caused by somatic mosaic mutations of IDH1 and
IDH2, Nat. Genet. 43 (2011) 1262e1265.
[11] K. Ichimura, Molecular pathogenesis of IDH mutations in gliomas, Brain Tumor Pathol. 29 (2012) 131e139.
[12] H. Arita, Y. Narita, A. Yoshida, N. Hashimoto, T. Yoshimine, K. Ichimura, IDH1/2
mutation detection in gliomas, Brain Tumor Pathol. 32 (2015) 79e89.
[13] P. Paschka, R.F. Schlenk, V.I. Gaidzik, M. Habdank, J. Kronke, L. Bullinger,
D. Spath, S. Kayser, M. Zucknick, K. Gotze, H.A. Horst, U. Germing, H. Dohner,
K. Dohner, IDH1 and IDH2 mutations are frequent genetic alterations in acute
myeloid leukemia and confer adverse prognosis in cytogenetically normal
acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem
duplication, J. Clin. Oncol. 28 (2010) 3636e3643.
[14] K.E. Yen, M.A. Bittinger, S.M. Su, V.R. Fantin, Cancer-associated IDH mutations:biomarker and therapeutic opportunities, Oncogene 29 (2010) 6409e6417.
[15] L. Sellner, D. Capper, J. Meyer, C.D. Langhans, C.M. Hartog, H. Pfeifer, H. Serve,

Y. Fujii et al. / Biochemical and Biophysical Research Communications 466 (2015) 733e739
A.D. Ho, J.G. Okun, A. Kramer, A. Von Deimling, Increased levels of 2hydroxyglutarate in AML patients with IDH1-R132H and IDH2-R140Q mutations, Eur. J. Haematol. 85 (2010) 457e459.
[16] M. Kato Kaneko, S. Ogasawara, Y. Kato, Establishment of a multi-speciﬁc
monoclonal antibody MsMab-1 recognizing both IDH1 and IDH2 mutations,
Tohoku J. Exp. Med. 230 (2013) 103e109.
[17] X. Liu, Y. Kato, M.K. Kaneko, M. Sugawara, S. Ogasawara, Y. Tsujimoto,Y. Naganuma, M. Yamakawa, T. Tsuchiya, M. Takagi, Isocitrate dehydrogenase
2 mutation is a frequent event in osteosarcoma detected by a multi-speciﬁc
monoclonal antibody MsMab-1, Cancer Med. 2 (2013) 803e814.
[18] S. Takano, Y. Kato, T. Yamamoto, M.K. Kaneko, E. Ishikawa, Y. Tsujimoto,
M. Matsuda, K. Nakai, R. Yanagiya, S. Morita, K. Tsuboi, A. Matsumura,
Immunohistochemical detection of IDH1 mutation, p53, and internexin as
prognostic factors of glial tumors, J. Neurooncol 108 (2012) 361e373.
[19] S. Ogasawara, M.K. Kaneko, Y. Tsujimoto, X. Liu, Y. Kato, Multi-speciﬁc
monoclonal antibody MsMab-2 recognizes IDH1-R132L and IDH2-R172M
mutations, Monoclon. Antib. Immunodiagn. Immunother. 32 (2013) 377e381.
[20] 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 reactingwith its platelet-aggregation-stimulating domain, Biochem. Biophys. Res.
Commun. 349 (2006) 1301e1307.
[21] 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 a dodecapeptide derived from human podoplanin, Protein Expr. Purif. 95 (2014)240e247.
[22] Y. Kato, M.K. Kaneko, A cancer-speciﬁc monoclonal antibody recognizes theaberrantly glycosylated podoplanin, Sci. Rep. 4 (2014) 5924.
[23] M.K. Kaneko, H. Oki, S. Ogasawara, M. Takagi, Y. Kato, Anti-podoplanin
Monoclonal antibody LpMab-7 detects metastatic lesions of osteosarcoma,
Monoclon. Antib. Immunodiagn. Immunother. 34 (2015) 154e161.
[24] Y. Kato, Speciﬁc monoclonal antibodies against IDH1/2 mutations as diagnostic tools for gliomas, Brain Tumor Pathol. 32 (2015) 3e11.
[25] S. Takano, Y. Kato, T. Yamamoto, X. Liu, E. Ishikawa, M.K. Kaneko,
S. Ogasawara, M. Matsuda, M. Noguchi, A. Matsumura, Diagnostic advantage739

of double immunohistochemistry using two mutation-speciﬁc anti-IDH antibodies (HMab-1 and MsMab-1) in gliomas, Brain Tumor Pathol. 32 (2015)
169e175.
[26] H. Arita, Y. Narita, A. Yoshida, N. Hashimoto, T. Yoshimine, K. Ichimura, IDH1/2
mutation detection in gliomas, Brain Tumor Pathol. 32 (2015) 79e89.
[27] B. Pang, M.B. Durso, R.L. Hamilton, M.N. Nikiforova, A novel COLD-PCR/FMCA
assay enhances the detection of low-abundance IDH1 mutations in gliomas,
Diagn Mol. Pathol. 22 (2013) 28e34.
[28] H. Arita, Y. Narita, Y. Matsushita, S. Fukushima, A. Yoshida, H. Takami,
Y. Miyakita, M. Ohno, S. Shibui, K. Ichimura, Development of a robust andsensitive pyrosequencing assay for the detection of IDH1/2 mutations in gliomas, Brain Tumor Pathol. 32 (2015) 22e30.
[29] O.C. Andronesi, G.S. Kim, E. Gerstner, T. Batchelor, A.A. Tzika, V.R. Fantin,
M.G. Vander Heiden, A.G. Sorensen, Detection of 2-hydroxyglutarate in IDHmutated glioma patients by in vivo spectral-editing and 2D correlation
magnetic resonance spectroscopy, Sci. Transl. Med. 4 (2012), 116ra114.
[30] Y. Kato, G. Jin, C.T. Kuan, R.E. McLendon, H. Yan, D.D. Bigner, A monoclonal
antibody IMab-1 speciﬁcally recognizes IDH1R132H, the most common
glioma-derived mutation, Biochem. Biophys. Res. Commun. 390 (2009)
547e551.
[31] M.K. Kaneko, W. Tian, S. Takano, H. Suzuki, Y. Sawa, Y. Hozumi, K. Goto,
K. Yamazaki, C. Kitanaka, Y. Kato, Establishment of a novel monoclonal antibody SMab-1 speciﬁc for IDH1-R132S mutation, Biochem. Biophys. Res.
Commun. 406 (2011) 608e613.
[32] M.K. Kaneko, S. Morita, Y. Tsujimoto, R. Yanagiya, K. Nasu, H. Sasaki,
Y. Hozumi, K. Goto, A. Natsume, M. Watanabe, T. Kumabe, S. Takano, Y. Kato,
Establishment of novel monoclonal antibodies KMab-1 and MMab-1 speciﬁc
for IDH2 mutations, Biochem. Biophys. Res. Commun. 432 (2013) 40e45.
[33] Y. Kato, M.K. Kaneko, Generation of a novel monoclonal antibody WMab-1
speciﬁc for IDH2-R172W mutation, Biochem. Biophys. Res. Commun. 433(2013) 374e378.
[34] D. Capper, H. Zentgraf, J. Balss, C. Hartmann, A. von Deimling, Monoclonal
antibody speciﬁc for IDH1 R132H mutation, Acta Neuropathol. 118 (2009)599e601.